Polynucleotide agents targeting aminolevulinic acid synthase-1 (alas1) and uses thereof

ABSTRACT

The invention relates to polynucleotide agents, e.g., antisense polynucleotide agents, targeting the ALAS1 gene, and methods of using such agents to alter (e.g., inhibit) expression of ALAS1 and to treat ALAS1 associated diseases, e.g., porphyria.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/473,836, filed on Mar. 30, 20217, which is a 35 § U.S.C. 111(a)continuation application which claims the benefit of priority toPCT/US2015/055989, filed on Oct. 16, 2015, which claims priority to U.S.Provisional Application No. 62/065,293, filed on Oct. 17, 2014. Theentire contents of each of the foregoing applications are incorporatedherein by reference.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Feb. 16, 2021, is named121301_02603_SL.txt and is 348,527 bytes in size.

BACKGROUND OF THE INVENTION

The inherited porphyrias are a family of disorders resulting from thedeficient activity of specific enzymes in the heme biosynthetic pathway,also referred to herein as the porphyrin pathway. Deficiency in theenzymes of the porphyrin pathway leads to insufficient heme productionand to an accumulation of porphyrin precursors and porphyrins, which aretoxic to tissue in high concentrations.

Of the inherited porphyrias, acute intermittent porphyria (ATP, e.g.,autosomal dominant AIP), variegate porphyria (VP, e.g., autosomaldominant VP), hereditary coproporphyria (copropophyria or HCP, e.g.,autosomal dominant HCP), and 5′ aminolevulinic acid (also known asS-aminolevulinic acid or ALA) dehydratase deficiency porphyria (ADP,e.g., autosomal recessive ADP) are classified as acute hepaticporphyrias and are manifested by acute neurological attacks that can belife threatening. The acute attacks are characterized by autonomic,peripheral, and central nervous system symptoms, including severeabdominal pain, hypertension, tachycardias, constipation, motorweakness, paralysis, and seizures. If not treated properly,quadriplegia, respiratory impairment, and death may ensue. Variousfactors, including cytrochrome P450-inducing drugs, dieting, andhormonal changes can precipitate acute attacks by increasing theactivity of hepatic 5′-aminolevulinic acid synthase 1 (ALAS1), the firstand rate-limiting enzyme of the heme biosynthetic pathway. In the acuteporphyrias, e.g., AIP, VP, HCP and ADP, the respective enzymedeficiencies result in hepatic production and accumulation of one ormore substances (e.g., porphyrins and/or porphyrin precursors, e.g., ALAand/or PBG (porphobilinogen)) that can be neurotoxic and can result inthe occurrence of acute neurologic attacks. See, e.g., Balwani, M andDesnick, R. J., Blood, 120:4496-4504, 2012.

The current therapy for acute neurologic attacks is the intravenousadministration of hemin (Panhematin®, Lundbeck or Normosang®, OrphanEurope), which provides exogenous heme for the negative feedbackinhibition of ALAS1 and, thereby, decreases production of ALA and PBG.Hemin is used for treatment during an acute attack and for prevention ofattacks, particularly in women with acute porphyrias who experiencefrequent attacks with hormonal changes during their menstrual cycles.While patients generally respond well, its effect is slow, typicallytaking two to four days or longer to normalize urinary ALA and PBGconcentrations towards normal levels. As the intravenous hemin israpidly metabolized, three to four infusions are usually necessary toeffectively treat or prevent an acute attack. In addition, repeatedinfusions may cause iron overload and phlebitis, which may compromiseperipheral venous access. Although orthotrophic liver transplantation iscurative, this procedure is associated with significant morbidity andmortality and the availability of liver donors is limited. Therefore, analternative therapeutic approach that is more effective, fast-acting,and safe is needed. It would be particularly advantageous if suchtreatment could be delivered by subcutaneous administration, as thiswould preclude the need for infusions and prolonged hospitalization.

SUMMARY OF THE INVENTION

The present invention provides polynucleotide agents and compositionscomprising such agents which target nucleic acids encoding5′-aminolevulinic acid synthase 1 (ALAS1) and interfere with the normalfunction of the targeted nucleic acid. The ALAS1 nucleic acid may bewithin a cell, e.g., a cell within a subject, such as a human. Thepresent invention also provides methods and combination therapies fortreating a subject having a disorder that would benefit from inhibitingor reducing the expression of an ALAS1 mRNA, e.g., an ALAS1-associateddisease, e.g., a porphyria, e.g., acute intermittent porphyria (ATP)porphyria and ALA dehydratase deficiency porphyria (ADP), using thepolynucleotide agents and compositions of the invention.

Accordingly, in one aspect, the present invention provides an antisensepolynucleotide agent for inhibiting expression of an aminolevulinic acidsynthase-1 (ALAS1) gene, wherein the agent comprises about 4 to about 50contiguous nucleotides, wherein at least one of the contiguousnucleotides is a modified nucleotide, and wherein the nucleotidesequence of the agent is about 80% complementary over its entire lengthto the equivalent region of the nucleotide sequence of any one of SEQ IDNOs:1-2.

In one embodiment, the equivalent region is any one of the targetregions of SEQ ID NO:1 provided in Tables 3 and 4.

In one aspect, the invention provides an antisense polynucleotide agentfor inhibiting expression of an aminolevulinic acid synthase-1 (ALAS1)gene, wherein the agent comprises at least 8 contiguous nucleotidesdiffering by no more than 3 nucleotides from any one of the nucleotidesequences listed in Tables 3 and 4.

In one embodiment, substantially all of the nucleotides of the antisensepolynucleotide agent are modified nucleotides.

In another embodiment, all of the nucleotides of the antisensepolynucleotide agent are modified nucleotides.

The agent may be 10 to 40 nucleotides in length, 10 to 30 nucleotides inlength, 18 to 30 nucleotides in length, 10 to 24 nucleotides in length,18 to 24 nucleotides in length, 21 nucleotides in length, or 20nucleotides in length.

In some embodiments, the modified nucleotide comprises a modified sugarmoiety selected from the group consisting of: a 2′-O-methoxyethylmodified sugar moiety, a 2′-methoxy modified sugar moiety, a 2′-O-alkylmodified sugar moiety, and a bicyclic sugar moiety.

sugar moiety, a 2′-O-alkyl modified sugar moiety, and a bicyclic sugarmoiety.

In one embodiment, the bicyclic sugar moiety has a (—CRH-)n groupforming a bridge between the 2′ oxygen and the 4′ carbon atoms of thesugar ring, wherein n is 1 or 2 and wherein R is H, CH₃ or CH₃OCH₃.

In a further embodiment, n is 1 and R is CH₃.

In another embodiment, the modified nucleotide is a 5-methylcytosine.

In another embodiment, the modified nucleotide includes a modifiedinternucleoside linkage, such as a phosphorothioate internucleosidelinkage.

In one embodiment, an agent of the invention comprises one2′-deoxynucleotide. In another embodiment, an agent of the inventioncomprises one 2′-deoxynucleotide flanked on each side by at least onenucleotide having a modified sugar moiety.

In one embodiment, an agent of the invention comprises a plurality,e.g., more than 1, e.g., 2, 3, 4, 5, 6, or 7, 2′-deoxynucleotides. Inone embodiment, an agent of the invention comprises a plurality, e.g.,more than 1, 2′-deoxynucleotides flanked on each side by at least onenucleotide having a modified sugar moiety.

In one embodiment, the agent is a gapmer comprising a gap segmentcomprised of linked 2′-deoxynucleotides positioned between a 5′ and a 3′wing segment.

In one embodiment, the modified sugar moiety is selected from the groupconsisting of a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxymodified sugar moiety, a 2′-O-alkyl modified sugar moiety, and abicyclic sugar moiety.

In one embodiment, the agent including about 4 to about 50 contiguousnucleotides includes a plurality of 2′-deoxynucleotides flanked on eachside by at least one nucleotide having a modified sugar moiety.

In one embodiment, the 5′-wing segment is 1 to 6 nucleotides in length.

In one embodiment, the 3′-wing segment is 1 to 6 nucleotides in length.

In one embodiment, the gap segment is 5 to 14 nucleotides in length.

In one embodiment, the 5′-wing segment is 6 nucleotides in length.

In one embodiment, the 3′-wing segment is 6 nucleotides in length.

In one embodiment, the 5′-wing segment is 5 nucleotides in length.

In one embodiment, the 3′-wing segment is 5 nucleotides in length.

In one embodiment, the 5′-wing segment is 4 nucleotides in length.

In one embodiment, the 3′-wing segment is 4 nucleotides in length.

In one embodiment, the 5′-wing segment is 3 nucleotides in length.

In one embodiment, the 3′-wing segment is 3 nucleotides in length.

In one embodiment, gap segment is 10 nucleotides in length.

In one embodiment, gap segment is 11 nucleotides in length.

In another aspect, the invention provides an antisense polynucleotideagent for inhibiting aminolevulinic acid synthase-1 (ALAS1) expression,including a gap segment consisting of linked deoxynucleotides; a 5′-wingsegment consisting of linked nucleotides; a 3′-wing segment consistingof linked nucleotides; such that the gap segment is positioned betweenthe 5′-wing segment and the 3′-wing segment and wherein each nucleotideof each wing segment comprises a modified sugar.

In one embodiment, the gap segment is ten 2′-deoxynucleotides in lengthand each of the wing segments is 5 nucleotides in length.

In another embodiment, the gap segment is eleven 2′-deoxynucleotides inlength and each of the wing segments is 5 nucleotides in length.

In yet another embodiment, the gap segment is ten 2′-deoxynucleotides inlength and each of the wing segments is 4 nucleotides in length.

In some embodiments, the gap segment is eleven 2′-deoxynucleotides inlength and each of the wing segments is 4 nucleotides in length.

In one embodiment, the gap segment is ten 2′-deoxynucleotides in lengthand each of the wing segments is 3 nucleotides in length.

In one embodiment, the gap segment is eleven 2′-deoxynucleotides inlength and each of the wing segments is 3 nucleotides in length.

In one embodiment, the gap segment is ten 2′-deoxynucleotides in lengthand each of the wing segments is 2 nucleotides in length.

In one embodiment, the gap segment is eleven 2′-deoxynucleotides inlength and each of the wing segments is 2 nucleotides in length.

In one embodiment, the modified sugar moiety of the agent for inhibitingaminolevulinic acid synthase-1 (ALAS1) expression is selected from thegroup consisting of a 2′-O-methoxyethyl modified sugar moiety, a2′-methoxy modified sugar moiety, a 2′-O-alkyl modified sugar moiety,and a bicyclic sugar moiety.

In yet another aspect of the invention, the polynucleotide agent forinhibiting expression of aminolevulinic acid synthase-1 (ALAS1) furtherincludes a ligand.

In one embodiment, the antisense polynucleotide agent is conjugated tothe ligand at the 3′-terminus.

In one embodiment the ligand is an N-acetylgalactosamine (GalNAc)derivative.

For example, the ligand is:

Further, in another aspect, the invention provides a pharmaceuticalcomposition for inhibiting expression of a aminolevulinic acidsynthase-1 (ALAS1) gene including an antisense polynucleotide forinhibiting ALAS1 expression as described herein.

In one embodiment, the agent is present in an unbuffered solution.

In one embodiment, the unbuffered solution is saline or water.

In another embodiment, the agent is present in a buffer solution.

In one embodiment, the buffer solution includes acetate, citrate,prolamine, carbonate, or phosphate or any combination thereof.

In one embodiment, the buffer solution is phosphate buffered saline(PBS).

In one embodiment, the pharmaceutical composition includes a lipidformulation.

In one embodiment, the lipid formulation includes a LNP.

In another embodiment, the lipid formulation includes a MC3.

In another aspect, the invention provides a method of inhibitingaminolevulinic acid synthase-1 (ALAS1) expression in a cell, the methodincluding contacting the cell with any one of the agents orpharmaceutical compositions described herein; and maintaining the cellproduced in step (a) for a time sufficient to obtain antisenseinhibition of an ALAS1 gene, thereby inhibiting expression of the ALASgene in the cell.

In one embodiment, the cell is within a subject.

In one embodiment, the subject is a human.

In one embodiment, the ALAS1 expression is inhibited by at least about30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, about 98% or about 100%.

In yet another aspect, the invention provides a method of treating asubject having a disease or disorder that would benefit from reductionin aminolevulinic acid synthase-1 (ALAS1) expression, the methodincluding administering to the subject a therapeutically effectiveamount of any one of the agents or the pharmaceutical compositionsdescribed above, thereby treating the subject.

In another aspect, the invention provides a method of preventing atleast one symptom in a subject having a disease or disorder that wouldbenefit from reduction in aminolevulinic acid synthase-1 (ALAS1)expression, the method including administering to the subject aprophylactically effective amount of any one of the agents or thepharmaceutical compositions described above, thereby preventing at leastone symptom in the subject having a disorder that would benefit fromreduction in ALAS1 expression.

In one embodiment, the administration of the antisense polynucleotideagent to the subject causes a decrease in ALAS1 protein levels.

In one embodiment, the disorder is an ALAS1-associated disease.

For example, the ALAS1-associated disease is porphyria, e.g., theporphyria is one of: X-linked sideroblastic anemia (XLSA), ALAdeyhdratase deficiency porphyria (Doss porphyria), acute intermittentporphyria (AIP), congenital erythropoietic porphyria (CEP), prophyriacutanea tarda (PCT), hereditary coproporphyria (coproporphyria, or HCP),variegate porphyria (VP), erythropoietic protoporphyria (EPP), ortransient erythroporphyria of infancy, acute hepatic porphyria,hepatoerythropoietic porphyria, or dual porphyria.

In one embodiment, an ALAS1-associated disease, is a hepatic porphyria,e.g., a hepatic porphyria characterized by a deficiency in the enzymeporphobilinogen deaminase (PBGD), such as acute intermittent porphyria(AIP) porphyria. In another embodiment, an ALAS1-associated disease, isa hepatic porphyria, e.g., a hepatic porphyria characterized byoverexpression of δ-aminolevulinic acid synthase 1 (ALAS1) in the liver,such as ALA dehydratase deficiency porphyria (ADP).

In one embodiment, the agent or the composition is administered after anacute attack of porphyria.

In another embodiment, the agent or the composition is administeredduring an acute attack of porphyria.

In one embodiment, the agent or composition is administeredprophylactically to prevent an acute attack of porphyria.

In one embodiment, the subject is human.

In one embodiment, the agent is administered at a dose of about 0.01mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg.

In one embodiment, the agent is administered at a dose of about 10 mg/kgto about 30 mg/kg.

In one embodiment, the agent is administered to the subject once a week.

In one embodiment, the agent is administered to the subject twice aweek.

In one embodiment, the agent is administered to the subject twice amonth.

In one embodiment, the agent is administered to the subjectsubcutaneously.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the heme biosynthetic pathway.

FIG. 2 summarizes certain porphyrias associated with genetic errors inheme metabolism.

FIG. 3 depicts a human ALAS1 mRNA sequence transcript variant 1 (Ref.Seq. NM_000688.4 (GI:40316942, record dated Nov. 19, 2011), SEQ ID NO:1).

FIG. 4 depicts a human ALAS1 mRNA sequence transcript variant 2 (Ref.Seq. NM_000688.5 (GI: 362999011, record dated Apr. 1, 2012), SEQ ID NO:2).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides polynucleotide agents and compositionscomprising such agents which target nucleic acids encoding ALAS1 (e.g.,mRNA encoding ALAS1 as provided in, for example, any one of SEQ ID NO:1(NM_000688.4) or SEQ ID NO:2 (NM_000688.5)). The polynucleotide agentsbind to nucleic acids encoding SEQ ID NO:1 via, e.g., Watson-Crick basepairing, and interfere with the normal function of the targeted nucleicacid.

The polynucleotide agents of the invention include a nucleotide sequencewhich is about 4 to about 50 nucleotides or less in length and which isabout 80% complementary to at least part of an mRNA transcript of anALAS1 gene. The use of these polynucleotide agents enables the targetedinhibition of RNA expression and/or activity of an ALAS1 gene inmammals.

The present inventors have demonstrated that polynucleotide agentstargeting ALAS1 can mediate antisense inhibition in vitro resulting insignificant inhibition of expression of an ALAS1 gene. Thus, methods andcompositions including these polynucleotide agents are useful fortreating a subject who would benefit by a reduction in the levels and/oractivity of an ALAS1 protein, such as a subject having anALAS1-associated disease, e.g., a porphyria.

The present invention also provides methods and combination therapiesfor treating a subject having a disorder that would benefit frominhibiting or reducing the expression of an ALAS1 gene, such as anALAS1-associated disease, e.g., a porphyria, using the polynucleotideagents and compositions of the invention.

The present invention also provides methods for preventing at least onesymptom, e.g., severe abdominal pain, in a subject having a disorderthat would benefit from inhibiting or reducing the expression of anALAS1 gene, e.g., an ALAS1-associated disease, e.g., a porphyria. Thepresent invention further provides compositions comprisingpolynucleotide agents which effect antisense inhibition of an ALAS1gene. The ALAS1 gene may be within a cell, e.g., a cell within asubject, such as a human.

The combination therapies of the present invention include administeringto a subject having an ALAS1-associated disease, a polynucleotide agentof the invention and an additional therapeutic, such as glucose and/or aheme product such as hemin. The combination therapies of the inventionreduce ALAS1 levels in the subject (e.g., by about 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 99%) bytargeting ALAS1 mRNA with a polynucleotide agent of the invention and,accordingly, allow the therapeutically (or prophylactically) effectiveamount of the additional therapeutic required to treat the subject to bereduced, thereby decreasing the costs of treatment and permitting easierand more convenient ways of administering the additional therapeutic,such as subcutaneous administration.

The following detailed description discloses how to make and usepolynucleotide agents to inhibit the mRNA and/or protein expression ofan ALAS1 gene, as well as compositions, uses, and methods for treatingsubjects having diseases and disorders that would benefit frominhibition and/or reduction of the expression of this gene.

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

As used herein, “ALAS1” (also known as ALAS1; S-aminolevulinate synthase1; 6-ALA synthase 1; 5′-aminolevulinic acid synthase 1; ALAS-H; ALASH;ALAS-N; ALAS3; EC2.3.1.37; 5-aminolevulinate synthase, nonspecific,mitochondrial; ALAS; MIG4; OTTHUMP00000212619; OTTHUMP00000212620;OTTHUMP00000212621; OTTTHUMP00000212622; migration-inducing protein 4;EC 2.3.1) refers to a nuclear-encoded mitochondrial enzyme that is thefirst and rate-limiting enzyme in the mammalian heme biosyntheticpathway. ALAS1 catalyzes the condensation of glycine with succinyl-CoAto form S-aminolevulinic acid (ALA). The level of the mature encodedALAS1 protein is regulated by heme: high levels of heme down-regulatethe mature enzyme in mitochondria while low heme levels up-regulate.Multiple alternatively spliced variants, encoding the same protein, havebeen identified.

The human ALAS1 gene is expressed ubiquitously, is found on chromosome3p21.1 and typically encodes a sequence of 640 amino acids. In contrast,the ALAS-2 gene, which encodes an isozyme, is expressed only inerythrocytes, is found on chromoxome Xp11.21, and typically encodes asequence of 550 amino acids.

As used herein an “ALAS1 protein” means any protein variant of ALAS1from any species (e.g., human, mouse, non-human primate), as well as anymutants and fragments thereof that retain an ALAS1 activity. Similarly,an “ALAS1 transcript” refers to any transcript variant of ALAS1, fromany species (e.g., human, mouse, non-human primate). A sequence of ahuman ALAS1 variant 1 mRNA transcript can be found at NM_000688.4 (FIG.3; SEQ ID NO:1). Another version, a human ALAS1 variant 2 mRNAtranscript, can be found at NM_000688.5 (FIG. 4; SEQ ID NO:382).

Additional examples of ALAS1 mRNA sequences are readily available usingpublicly available databases, e.g., GenBank, Prosite, OMIM.

The term “ALAS1,” as used herein, also refers to naturally occurring DNAsequence variations of the ALAS1 gene, such as a single nucleotidepolymorphism in the ALAS1 gene (see, e.g., ncbi.nlm.nih.gov/snp).

The terms “antisense polynucleotide agent” “antisense compound”, and“agent” as used interchangeably herein, refer to an agent comprising asingle-stranded oligonucleotide that contains RNA as that term isdefined herein, and which targets nucleic acid molecules encoding ALAS1(e.g., mRNA encoding ALAS1 as provided in, for example, any one of SEQID NOs:1-2). The antisense polynucleotide agents specifically bind tothe target nucleic acid molecules via hydrogen bonding (e.g.,Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) andinterfere with the normal function of the targeted nucleic acid (e.g.,by an antisense mechanism of action). This interference with ormodulation of the function of a target nucleic acid by thepolynucleotide agents of the present invention is referred to as“antisense inhibition.”

The functions of the target nucleic acid molecule to be interfered withmay include functions such as, for example, translocation of the RNA tothe site of protein translation, translation of protein from the RNA,splicing of the RNA to yield one or more mRNA species, and catalyticactivity which may be engaged in or facilitated by the RNA.

In some embodiments, antisense inhibition refers to “inhibiting theexpression” of target nucleic acid levels and/or target protein levelsin a cell, e.g., a cell within a subject, such as a mammalian subject,in the presence of the antisense polynucleotide agent complementary to atarget nucleic acid as compared to target nucleic acid levels and/ortarget protein levels in the absence of the antisense polynucleotideagent. For example, the antisense polynucleotide agents of the inventioncan inhibit translation in a stoichiometric manner by base pairing tothe mRNA and physically obstructing the translation machinery, see Dias,N. et al., (2002) Mol Cancer Ther 1:347-355.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof an ALAS1 gene, including mRNA that is a product of RNA processing ofa primary transcription product.

As used herein, “target nucleic acid” refers to a nucleic acid moleculeto which an antisense polynucleotide agent specifically hybridizes.

As used herein, the term “specifically hybridizes” refers to anantisense polynucleotide agent having a sufficient degree ofcomplementarity between the antisense polynucleotide agent and a targetnucleic acid to induce a desired effect, while exhibiting minimal or noeffects on non-target nucleic acids under conditions in which specificbinding is desired, e.g., under physiological conditions in the case ofin vivo assays and therapeutic treatments.

A target sequence may be from about 4-50 nucleotides in length, e.g.,8-45, 10-45, 10-40, 10-35, 10-30, 10-20, 11-45, 11-40, 11-35, 11-30,11-20, 12-45, 12-40, 12-35, 12-30, 12-25, 12-20, 13-45, 13-40, 13-35,13-30, 13-25, 13-20, 14-45, 14-40, 14-35, 14-30, 14-25, 14-20, 15-45,15-40, 15-35, 15-30, 15-25, 15-20, 16-45, 16-40, 16-35, 16-30, 16-25,16-20, 17-45, 17-40, 17-35, 17-30, 17-25, 17-20, 18-45, 18-40, 18-35,18-30, 18-25, 18-20, 19-45, 19-40, 19-35, 19-30, 19-25, 19-20, e.g., 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, or 50 contiguous nucleotides of thenucleotide sequence of an mRNA molecule formed during the transcriptionof an ALAS1 gene. Ranges and lengths intermediate to the above recitedranges and lengths are also contemplated to be part of the invention.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” are used herein with respect to the base matching betweenan antisense polynucleotide agent and a target sequence. Theterm“complementarity” refers to the capacity for pairing betweennucleobases of a first nucleic acid and a second nucleic acid.

As used herein, an antisense polynucleotide agent that is “substantiallycomplementary to at least part of” a messenger RNA (mRNA) refers to anantisense polynucleotide agent that is substantially complementary to acontiguous portion of the mRNA of interest (e.g., an mRNA encodingALAS1). For example, a polynucleotide is complementary to at least apart of an ALAS1 mRNA if the sequence is substantially complementary toa non-interrupted portion of an mRNA encoding ALAS1.

As used herein, the term “region of complementarity” refers to theregion of the antisense polynucleotide agent that is substantiallycomplementary to a sequence, for example a target sequence, e.g., anALAS1 nucleotide sequence, as defined herein. Where the region ofcomplementarity is not fully complementary to the target sequence, themismatches can be in the internal or terminal regions of the molecule.Generally, the most tolerated mismatches are in the terminal regions,e.g., within 5, 4, 3, or 2 nucleotides of the 5′- and/or 3′-terminus ofthe antisense polynucleotide.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of apolynucleotide comprising the first nucleotide sequence to hybridize andform a duplex structure under certain conditions with the secondnucleotide sequence, as will be understood by the skilled person. Suchconditions can, for example, be stringent conditions, where stringentconditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C. or 70° C. for 12-16 hours followed by washing (see, e.g., “MolecularCloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring HarborLaboratory Press). Other conditions, such as physiologically relevantconditions as can be encountered inside an organism, can apply. Theskilled person will be able to determine the set of conditions mostappropriate for a test of complementarity of two sequences in accordancewith the ultimate application of the nucleotides.

Complementary sequences include those nucleotide sequences of anantisense polynucleotide agent of the invention that base-pair to asecond nucleotide sequence over the entire length of one or bothnucleotide sequences. Such sequences can be referred to as “fullycomplementary” with respect to each other herein. However, where a firstsequence is referred to as “substantially complementary” with respect toa second sequence herein, the two sequences can be fully complementary,or they can form one or more, but generally not more than 5, 4, 3 or 2mismatched base pairs upon hybridization for a duplex up to 30 basepairs, while retaining the ability to hybridize under the conditionsmost relevant to their ultimate application, e.g., antisense inhibitionof target gene expression.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, it will be understood that the terms“deoxyribonucleotide”, “ribonucleotide” and “nucleotide” can also referto a modified nucleotide, as further detailed below, or a surrogatereplacement moiety (see, e.g., Table 2). The skilled person is wellaware that guanine, cytosine, adenine, and uracil can be replaced byother moieties without substantially altering the base pairingproperties of an oligonucleotide comprising a nucleotide bearing suchreplacement moiety. For example, without limitation, a nucleotidecomprising inosine as its base can base pair with nucleotides containingadenine, cytosine, or uracil. Hence, nucleotides containing uracil,guanine, or adenine can be replaced in the nucleotide sequences of theagents featured in the invention by a nucleotide containing, forexample, inosine. In another example, adenine and cytosine anywhere inthe oligonucleotide can be replaced with guanine and uracil,respectively to form G-U Wobble base pairing with the target mRNA.Sequences containing such replacement moieties are suitable for thecompositions and methods featured in the invention.

A “nucleoside” is a base-sugar combination. The “nucleobase” (also knownas “base”) portion of the nucleoside is normally a heterocyclic basemoiety. “Nucleotides” are nucleosides that further include a phosphategroup covalently linked to the sugar portion of the nucleoside. Forthose nucleosides that include a pentofuranosyl sugar, the phosphategroup can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar.

“Polynucleotides,” also referred to as “oligonucleotides,” are formedthrough the covalent linkage of adjacent nucleosides to one another, toform a linear polymeric oligonucleotide. Within the polynucleotidestructure, the phosphate groups are commonly referred to as forming theinternucleoside linkages of the polynucleotide.

In general, the majority of nucleotides of the antisense polynucleotideagents are ribonucleotides, but as described in detail herein, theagents may also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide. In addition, as used in this specification, an“antisense polynucleotide agent” may include nucleotides (e.g.,ribonucleotides or deoxyribonucleotides) with chemical modifications; anantisense polynucleotide agent may include substantial modifications atmultiple nucleotides.

As used herein, the term “modified nucleotide” refers to a nucleotidehaving, independently, a modified sugar moiety, a modifiedinternucleotide linkage, and/or modified nucleobase. Thus, the termmodified nucleotide encompasses substitutions, additions or removal of,e.g., a functional group or atom, to internucleoside linkages, sugarmoieties, or nucleobases. The modifications suitable for use in theantisense polynucleiotde agents of the invention include all types ofmodifications disclosed herein or known in the art. Any suchmodifications, as used in nucleotides, are encompassed by “antisensepolynucleotide agent” for the purposes of this specification and claims.

As used herein, a “subject” is an animal, such as a mammal, including aprimate (such as a human, a non-human primate, e.g., a monkey, and achimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, ahorse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog,a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or agoose). In an embodiment, the subject is a human, such as a human beingtreated or assessed for a disease, disorder or condition that wouldbenefit from reduction in ALAS1 expression; a human at risk for adisease, disorder or condition that would benefit from reduction inALAS1 expression; a human having a disease, disorder or condition thatwould benefit from reduction in ALAS1 expression; and/or human beingtreated for a disease, disorder or condition that would benefit fromreduction in ALAS1 expression as described herein.

As used herein in the context of ALAS1 expression, the terms “treat,”“treating,” “treatment,” and the like, refer to relief from oralleviation of pathological processes related to ALAS1 expression (e.g.,pathological processes involving porphyrins or defects in the porphyrinpathway, such as, for example, porphyrias). In the context of thepresent invention insofar as it relates to any of the other conditionsrecited herein below (other than pathological processes related to ALAS1expression), the terms “treat,” “treatment,” and the like mean toprevent, relieve or alleviate at least one symptom associated with suchcondition, or to slow or reverse the progression or anticipatedprogression of such condition. For example, the methods featured herein,when employed to treat porphyria, may serve to reduce or prevent one ormore symptoms associated with porphyria (e.g., pain, vomiting,constipation, diarrhea, loss or impairment of movement, respiratoryparalysis, behavioral changes, including agitation, confusion,hallucinations, and depression, convulsions, as a result of excessivevomiting and/or diarrhea, and/or increased heart rate), to reduce theseverity or frequency of attacks associated with porphyria, to reducethe likelihood that an attack of one or more symptoms associated withporphyria will occur upon exposure to a precipitating condition, toshorten an attack associated with porphyria, and/or to reduce the riskof developing conditions associated with porphyria (e.g., kidney damage,hepatocellular cancer or neuropathy (e.g., progressive neuropathy).Thus, unless the context clearly indicates otherwise, the terms “treat,”“treatment,” and the like are intended to encompass prophylaxis, e.g.,prevention of disorders and/or symptoms of disorders related to ALAS1expression. “Treatment” can also mean prolonging survival as compared toexpected survival in the absence of treatment.

The term “lower” in the context of the level of an ALAS1 in a subject ora disease marker or symptom refers to a statistically significantdecrease in such level. The decrease can be, for example, at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or more and is preferably down to a levelaccepted as within the range of normal for an individual without suchdisorder.

As used herein, “prevention” or “preventing,” when used in reference toa disease, disorder or condition thereof, that would benefit from areduction in expression of an ALAS1 gene, refers to a reduction in thelikelihood that a subject will develop a symptom associated with such adisease, disorder, or condition, e.g., vomiting, constipation, diarrhea,loss or impairment of movement, respiratory paralysis, behavioralchanges, including agitation, confusion, hallucinations, and depression,convulsions, as a result of excessive vomiting and/or diarrhea,increased heart rate, and/or pain (e.g., neuropathic pain and/orneuropathy, e.g., progressive neuropathy). The failure to develop adisease, disorder or condition, or the reduction in the development of asymptom associated with such a disease, disorder or condition (e.g., byat least about 10% on a clinically accepted scale for that disease ordisorder), or the exhibition of delayed symptoms delayed (e.g., by days,weeks, months or years) is considered effective prevention.

II. Polynucleotide Agents of the Invention

The present invention provides polynucleotide agents, e.g., antisensepolynucleotide agents, and compositions comprising such agents, whichtarget an ALAS1 gene and inhibit the expression of the ALAS1 gene. Inone embodiment, the antisense polynucleotide agents inhibit theexpression of an ALAS1 gene in a cell, such as a cell within a subject,e.g., a mammal, such as a human having an ALAS1-associated disease,e.g., a porphyria, e.g., ATP or ADP.

The antisense polynucleotide agents of the invention include a region ofcomplementarity which is complementary to at least a part of an mRNAformed in the expression of an ALAS1 gene. The region of complementaritymay be about 50 nucleotides or less in length (e.g., about 50, 49, 48,47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30,29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, or 4 nucleotides or less in length). Upon contactwith a cell expressing the ALAS1 gene, the antisense polynucleotideagent inhibits the expression of the ALAS1 gene (e.g., a human, aprimate, a non-primate, or a bird ALAS1 gene) by at least about 10% asassayed by, for example, a PCR or branched DNA (bDNA)-based method, orby a protein-based method, such as by immunofluorescence analysis,using, for example, Western Blotting or flow cytometric techniques.

The region of complementarity between an antisense polynucleotide agentand a target sequence may be substantially complementary (e.g., there isa sufficient degree of complementarity between the antisensepolynucleotide agent and a target nucleic acid to so that theyspecifically hybridize and induce a desired effect), but is generallyfully complementary to the target sequence. The target sequence can bederived from the sequence of an mRNA formed during the expression of anALAS1 gene.

Accordingly, in one aspect, an antisense polynucleotide agent of theinvention specifically hybridizes to a target nucleic acid molecule,such as the mRNA encoding ALAS1, and comprises a contiguous nucleotidesequence which corresponds to the reverse complement of a nucleotidesequence of any one of SEQ ID NO:1-2, or a fragment of any one of SEQ IDNOs:1-2.

In some embodiments, the antisense polynucleotide agents of theinvention may be substantially complementary to the target sequence. Forexample, an antisense polynucleotide agent that is substantiallycomplementary to the target sequence may include a contiguous nucleotidesequence comprising no more than 5 mismatches (e.g., no more than 1, nomore than 2, no more than 3, no more than 4, or no more than 5mismatches) when hybridizing to a target sequence, such as to thecorresponding region of a nucleic acid which encodes a mammalian ALAS1mRNA. In some embodiments, the contiguous nucleotide sequence comprisesno more than a single mismatch when hybridizing to the target sequence,such as the corresponding region of a nucleic acid which encodes amammalian ALAS1 mRNA.

In some embodiments, the antisense polynucleotide agents of theinvention that are substantially complementary to the target sequencecomprise a contiguous nucleotide sequence which is at least about 80%complementary over its entire length to the equivalent region of thenucleotide sequence of any one of SEQ ID NOs:1-2, or a fragment of anyone of SEQ ID NOs:1-2, such as about 85%, about 86%, about 87%, about88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In some embodiments, an antisense polynucleotide agent comprises acontiguous nucleotide sequence which is fully complementary over itsentire length to the equivalent region of the nucleotide sequence of anyone of SEQ ID NOs:1-2 (or a fragment of any one of SEQ ID NOs:1-2).

An antisense polynucleotide agent may comprise a contiguous nucleotidesequence of about 4 to about 50 nucleotides in length, e.g., 8-49, 8-48,8-47, 8-46, 8-45, 8-44, 8-43, 8-42, 8-41, 8-40, 8-39, 8-38, 8-37, 8-36,8-35, 8-34, 8-33, 8-32, 8-31, 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24,8-23, 8-22, 8-21, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12,8-11, 8-10, 8-9, 10-49, 10-48, 10-47, 10-46, 10-45, 10-44, 10-43, 10-42,10-41, 10-40, 10-39, 10-38, 10-37, 10-36, 10-35, 10-34, 10-33, 10-32,10-31, 10-30, 10-29, 10-28, 10-27, 10-26, 10-25, 10-24, 10-23, 10-22,10-21, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12,10-11, 11-49, 11-48, 11-47, 11-46, 11-45, 11-44, 11-43, 11-42, 11-41,11-40, 11-39, 11-38, 11-37, 11-36, 11-35, 11-34, 11-33, 11-32, 11-31,11-30, 11-29, 11-28, 11-27, 11-26, 11-25, 11-24, 11-23, 11-22, 11-21,11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-49,12-48, 12-47, 12-46, 12-45, 12-44, 12-43, 12-42, 12-41, 12-40, 12-39,12-38, 12-37, 12-36, 12-35, 12-34, 12-33, 12-32, 12-31, 12-30, 12-29,12-28, 12-27, 12-26, 12-25, 12-24, 12-23, 12-22, 12-21, 12-20, 12-19,12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-49, 13-48, 13-47, 13-46,13-45, 13-44, 13-43, 13-42, 13-41, 13-40, 13-39, 13-38, 13-37, 13-36,13-35, 13-34, 13-33, 13-32, 13-31, 13-30, 13-29, 13-28, 13-27, 13-26,13-25, 13-24, 13-23, 13-22, 13-21, 13-20, 13-19, 13-18, 13-17, 13-16,13-15, 13-14, 14-49, 14-48, 14-47, 14-46, 14-45, 14-44, 14-43, 14-42,14-41, 14-40, 14-39, 14-38, 14-37, 14-36, 14-35, 14-34, 14-33, 14-32,14-31, 14-30, 14-29, 14-28, 14-27, 14-26, 14-25, 14-24, 14-23, 14-22,14-21, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-49, 15-48, 15-47,15-46, 15-45, 15-44, 15-43, 15-42, 15-41, 15-40, 15-39, 15-38, 15-37,15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27,15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17,15-16, 16-49, 16-48, 16-47, 16-46, 16-45, 16-44, 16-43, 16-42, 16-41,16-40, 16-39, 16-38, 16-37, 16-36, 16-35, 16-34, 16-33, 16-32, 16-31,16-30, 16-29, 16-28, 16-27, 16-26, 16-25, 16-24, 16-23, 16-22, 16-21,16-20, 16-19, 16-18, 16-17, 17-49, 17-48, 17-47, 17-46, 17-45, 17-44,17-43, 17-42, 17-41, 17-40, 17-39, 17-38, 17-37, 17-36, 17-35, 17-34,17-33, 17-32, 17-31, 17-30, 17-29, 17-28, 17-27, 17-26, 17-25, 17-24,17-23, 17-22, 17-21, 17-20, 17-19, 17-18, 18-49, 18-48, 18-47, 18-46,18-45, 18-44, 18-43, 18-42, 18-41, 18-40, 18-39, 18-38, 18-37, 18-36,18-35, 18-34, 18-33, 18-32, 18-31, 18-30, 18-29, 18-28, 18-27, 18-26,18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-49, 19-48, 19-47, 19-46,19-45, 19-44, 19-43, 19-42, 19-41, 19-40, 19-39, 19-38, 19-37, 19-36,19-35, 19-34, 19-33, 19-32, 19-31, 19-30, 19-29, 19-28, 19-27, 19-26,19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-49, 20-48, 20-47, 20-46,20-45, 20-44, 20-43, 20-42, 20-41, 20-40, 20-39, 20-38, 20-37, 20-36,20-35, 20-34, 20-33, 20-32, 20-31, 20-30, 20-29, 20-28, 20-27, 20-26,20-25, 20-24, 20-23, 20-22, 20-21, 21-49, 21-48, 21-47, 21-46, 21-45,21-44, 21-43, 21-42, 21-41, 21-40, 21-39, 21-38, 21-37, 21-36, 21-35,21-34, 21-33, 21-32, 21-31, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25,21-24, 21-23, 21-22, 22-49, 22-48, 22-47, 22-46, 22-45, 22-44, 22-43,22-42, 22-41, 22-40, 22-39, 22-38, 22-37, 22-36, 22-35, 22-34, 22-33,22-32, 22-31, 22-30, 22-29, 22-28, 22-27, 22-26, 22-25, 22-24, 22-23,23-49, 23-48, 23-47, 23-46, 23-45, 23-44, 23-43, 23-42, 23-41, 23-40,23-39, 23-38, 23-37, 23-36, 23-35, 23-34, 23-33, 23-32, 23-31, 23-30,23-29, 23-28, 23-27, 23-26, 23-25, 23-24, 24-49, 24-48, 24-47, 24-46,24-45, 24-44, 24-43, 24-42, 24-41, 24-40, 24-39, 24-38, 24-37, 24-36,24-35, 24-34, 24-33, 24-32, 24-31, 24-30, 24-29, 24-28, 24-27, 24-26,24-25, 25-49, 25-48, 25-47, 25-46, 25-45, 25-44, 25-43, 25-42, 25-41,25-40, 25-39, 25-38, 25-37, 25-36, 25-35, 25-34, 25-33, 25-32, 25-31,25-30, 25-29, 25-28, 25-27, 25-26, 26-49, 26-48, 26-47, 26-46, 26-45,26-44, 26-43, 26-42, 26-41, 26-40, 26-39, 26-38, 26-37, 26-36, 26-35,26-34, 26-33, 26-32, 26-31, 26-30, 26-29, 26-28, 26-27, 27-49, 27-48,27-47, 27-46, 27-45, 27-44, 27-43, 27-42, 27-41, 27-40, 27-39, 27-38,27-37, 27-36, 27-35, 27-34, 27-33, 27-32, 27-31, 27-30, 27-29, 27-28,28-49, 28-48, 28-47, 28-46, 28-45, 28-44, 28-43, 28-42, 28-41, 28-40,28-39, 28-38, 28-37, 28-36, 28-35, 28-34, 28-33, 28-32, 28-31, 28-30,28-29, 29-49, 29-48, 29-47, 29-46, 29-45, 29-44, 29-43, 29-42, 29-41,29-40, 29-39, 29-38, 29-37, 29-36, 29-35, 29-34, 29-33, 29-32, 29-31,29-30, 30-49, 30-48, 30-47, 30-46, 30-45, 30-44, 30-43, 30-42, 30-41,30-40, 30-39, 30-38, 30-37, 30-36, 30-35, 30-34, 30-33, 30-32, or 30-31nucleotides in length, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleotides in length.

In some embodiments, an antisense polynucleotide agent may comprise acontiguous nucleotide sequence of no more than 22 nucleotides, such asno more than 21 nucleotides, 20 nucleotides, 19 nucleotides, or no morethan 18 nucleotides. In some embodiments the antisense polynucleotideagents of the invention comprises less than 20 nucleotides. In otherembodiments, the antisense polynucleotide agents of the inventioncomprise 20 nucleotides.

In one aspect, an antisense polynucleotide agent of the inventionincludes a sequence selected from the group of sequences provided inTables 3 and 4. It will be understood that, although some of thesequences in Tables 3 and 4 are described as modified and/or conjugatedsequences, an antisense polynucleotide agent of the invention, may alsocomprise any one of the sequences set forth in Tables 3 and 4 that isun-modified, un-conjugated, and/or modified and/or conjugateddifferently than described therein.

By virtue of the nature of the nucleotide sequences provided in Tables 3and 4, antisense polynucleotide agents of the invention may include oneof the sequences of Tables 3 minus only a few nucleotides on one or bothends and yet remain similarly effective as compared to the antisensepolynucleotide agents described above. Hence, antisense polynucleotideagents having a sequence of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived fromone of the sequences of Tables 3 and 4 and differing in their ability toinhibit the expression of an ALAS1 gene by not more than about 5, 10,15, 20, 25, or 30% inhibition from an antisense polynucleotide agentcomprising the full sequence, are contemplated to be within the scope ofthe present invention.

In addition, the antisense polynucleotide agents provided in Tables 3and 4 identify a region(s) in an ALAS1 transcript that is susceptible toantisense inhibition (e.g., the regions encompassed by the start and endpositions relative to NM_000688.4 in Table 3 and NM_000688.5 in Table4). As such, the present invention further features antisensepolynucleotide agents that target within one of these sites. As usedherein, an antisense polynucleotide agent is said to target within aparticular site of an RNA transcript if the antisense polynucleotideagent promotes antisense inhibition of the target at that site. Such anantisense polynucleotide agent will generally include at least about 15contiguous nucleotides from one of the sequences provided in Tables 3and 4 coupled to additional nucleotide sequences taken from the regioncontiguous to the selected sequence in an ALAS1 gene.

While a target sequence is generally about 4-50 nucleotides in length,there is wide variation in the suitability of particular sequences inthis range for directing antisense inhibition of any given target RNA.Various software packages and the guidelines set out herein provideguidance for the identification of optimal target sequences for anygiven gene target, but an empirical approach can also be taken in whicha “window” or “mask” of a given size (as a non-limiting example, 20nucleotides) is literally or figuratively (including, e.g., in silico)placed on the target RNA sequence to identify sequences in the sizerange that can serve as target sequences. By moving the sequence“window” progressively one nucleotide upstream or downstream of aninitial target sequence location, the next potential target sequence canbe identified, until the complete set of possible sequences isidentified for any given target size selected. This process, coupledwith systematic synthesis and testing of the identified sequences (usingassays as described herein or as known in the art) to identify thosesequences that perform optimally can identify those RNA sequences that,when targeted with an antisense polynucleotide agent, mediate the bestinhibition of target gene expression. Thus, while the sequencesidentified, for example, in Tables 3 and 4 represent effective targetsequences, it is contemplated that further optimization of antisenseinhibition efficiency can be achieved by progressively “walking thewindow” one nucleotide upstream or downstream of the given sequences toidentify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., inTables 3 and 4, further optimization could be achieved by systematicallyeither adding or removing nucleotides to generate longer or shortersequences and testing those sequences generated by walking a window ofthe longer or shorter size up or down the target RNA from that point.Again, coupling this approach to generating new candidate targets withtesting for effectiveness of antisense polynucleotide agents based onthose target sequences in an inhibition assay as known in the art and/oras described herein can lead to further improvements in the efficiencyof inhibition. Further still, such optimized sequences can be adjustedby, e.g., the introduction of modified nucleotides as described hereinor as known in the art, addition or changes in length, or othermodifications as known in the art and/or discussed herein to furtheroptimize the molecule (e.g., increasing serum stability or circulatinghalf-life, increasing thermal stability, enhancing transmembranedelivery, targeting to a particular location or cell type, increasinginteraction with silencing pathway enzymes, increasing release fromendosomes) as an expression inhibitor.

III. Modified Polynucleotide Agents of the Invention

In one embodiment, the nucleotides of a polynucleotide agent of theinvention, e.g., an antisense polynucleotide agent of the invention, areun-modified, and do not comprise, e.g., chemical modifications and/orconjugations known in the art and described herein. In anotherembodiment, at least one of the nucleotides of a polynucleotide agent ofthe invention, e.g., an antisense polynucleotide agent of the invention,is chemically modified to enhance stability or other beneficialcharacteristics. In certain embodiments of the invention, substantiallyall of the nucleotides of a polynucleotide agent of the invention, e.g.,an antisense polynucleotide agent of the invention, are modified. Inother embodiments of the invention, all of the nucleotides of apolynucleotide agent of the invention, e.g., an antisense polynucleotideagent of the invention, are modified. Antisense polynucleotide agents ofthe invention in which “substantially all of the nucleotides aremodified” are largely but not wholly modified and can include not morethan 5, 4, 3, 2, or 1 unmodified nucleotides.

The nucleic acids featured in the invention can be synthesized and/ormodified by standard methods known in the art as further discussedbelow, e.g., solution-phase or solid-phase organic synthesis or both,e.g., by use of an automated DNA synthesizer, such as are commerciallyavailable from, for example, Biosearch, Applied Biosystems, Inc.Well-established methods for the synthesis and/or modification of thenucleic acids featured in the invention are described in, for example,“Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Modifications include, for example,end modifications, e.g., 5′-end modifications (phosphorylation,conjugation, inverted linkages) or 3′-end modifications (conjugation,DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases; sugar modifications (e.g., atthe 2′-position or 4′-position) or replacement of the sugar; and/orbackbone modifications, including modification or replacement of thephosphodiester linkages.

Specific examples of modified nucleotides useful in the embodimentsdescribed herein include, but are not limited to nucleotides containingmodified backbones or no natural internucleoside linkages. Nucleotideshaving modified backbones include, among others, those that do not havea phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modifiednucleotides that do not have a phosphorus atom in their internucleosidebackbone can also be considered to be oligonucleosides. In someembodiments, a modified antisense polynucleotide agent will have aphosphorus atom in its internucleoside backbone.

Modified nucleotide backbones include, for example, phosphorothioates,chiral phosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. No.RE39,464, the entire contents of each of which are hereby incorporatedherein by reference.

Modified nucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatoms and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

In other embodiments, suitable nucleotide mimetics are contemplated foruse in antisense polynucleotide agents, in which both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an RNA mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar backbone of an RNA is replacedwith an amide containing backbone, in particular an aminoethylglycinebackbone. The nucleobases are retained and are bound directly orindirectly to aza nitrogen atoms of the amide portion of the backbone.Representative U.S. patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, the entire contents of each of which are herebyincorporated herein by reference. Additional PNA compounds suitable foruse in the antisense polynucleotide agents of the invention aredescribed in, for example, in Nielsen et al., Science, 1991, 254,1497-1500.

Some embodiments featured in the invention include polynucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, theantisense polynucleotide agents featured herein have morpholino backbonestructures of the above-referenced U.S. Pat. No. 5,034,506.

Modified nucleotides can also contain one or more modified orsubstituted sugar moieties. The antisense polynucleotide agents featuredherein can include one of the following at the 2′-position: OH; F; O-,S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Exemplary suitable modifications include O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.

In other embodiments, antisense polynucleotide agents include one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br,CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an antisense polynucleotide,or a group for improving the pharmacodynamic properties of an antisensepolynucleotide agent, and other substituents having similar properties.In some embodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on a nucleotide of an antisensepolynucleotide agent, particularly the 3′ position of the sugar on the3′ terminal nucleotide. Antisense polynucleotide agents can also havesugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application. The entire contents of eachof the foregoing are hereby incorporated herein by reference.

Additional nucleotides having modified or substituted sugar moieties foruse in the polynucleotide agents of the invention include nucleotidescomprising a bicyclic sugar. A “bicyclic sugar” is a furanosyl ringmodified by the bridging of two atoms. A“bicyclic nucleoside” (“BNA”) isa nucleoside having a sugar moiety comprising a bridge connecting twocarbon atoms of the sugar ring, thereby forming a bicyclic ring system.In certain embodiments, the bridge connects the 4′-carbon and the2′-carbon of the sugar ring. Thus, in some embodiments an antisensepolynucleotide agent may include one or more locked nucleic acids. A“locked nucleic acid” (“LNA”) is a nucleotide having a modified ribosemoiety in which the ribose moiety comprises an extra bridge connectingthe 2′ and 4′ carbons. In other words, an LNA is a nucleotide comprisinga bicyclic sugar moiety comprising a 4′-CH₂—O-2′ bridge. This structureeffectively “locks” the ribose in the 3′-endo structural conformation.The addition of locked nucleic acids to santisense polynucleotide agentshas been shown to increase santisense polynucleotide agent stability inserum, and to reduce off-target effects (Elmen, J. et al., (2005)Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol CancTher 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research31(12):3185-3193).

Examples of bicyclic nucleosides for use in the polynucleotides of theinvention include without limitation nucleosides comprising a bridgebetween the 4′ and the 2′ ribosyl ring atoms. In certain embodiments,the antisense polynucleotide agents of the invention include one or morebicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′to 2′ bridged bicyclic nucleosides, include but are not limited to4′-(CH2)-O-2′ (LNA); 4′-(CH2)S-2′; 4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3) 0-2′(also referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH2OCH3)0-2′ (and analogs thereof, see, e.g., U.S. Pat. No. 7,399,845);4′-C(CH3)(CH3) 0-2′ (and analogs thereof; see e.g., U.S. Pat. No.8,278,283); 4′-CH2-N(OCH3)-2′ (and analogs thereof, see e.g., U.S. Pat.No. 8,278,425); 4′-CH2 O—N(CH3)-2′ (see, e.g., U.S. Patent PublicationNo. 2004/0171570); 4′-CH2-N(R)—O-2′, wherein R is H, C1-C12 alkyl, or aprotecting group (see, e.g., U.S. Pat. No. 7,427,672);4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya et al., J Org. Chem.,2009, 74, 118-134); and 4′-CH2-C(═CH2)-2′ (and analogs thereof, see,e.g., U.S. Pat. No. 8,278,426). The entire contents of each of theforegoing are hereby incorporated herein by reference.

Additional representative U.S. Patents and US Patent Publications thatteach the preparation of locked nucleic acid nucleotides include, butare not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191;6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133;7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193;8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US2009/0012281, the entire contents of each of which are herebyincorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one ormore stereochemical sugar configurations including for exampleα-L-ribofuranose and j-D-ribofuranose (see WO 99/14226).

In one particular embodiment of the invention, an antisensepolynucleotide agent can include one or more constrained ethylnucleotides. As used herein, a “constrained ethyl nucleotide” or “cEt”is a locked nucleic acid comprising a bicyclic sugar moiety comprising a4′-CH(CH₃)—O-2′ bridge. In one embodiment, a constrained ethylnucleotide is in an S conformation and is referred to as an“S-constrained ethyl nucleotide” or “S-cEt.”

Modified nucleotides included in the antisense polynucleotide agents ofthe invention can also contain one or more sugar mimetics. For example,the antisense polynucleotide agent may include a “modifiedtetrahydropyran nucleotide” or “modified THP nucleotide.” A “modifiedtetrahydropyran nucleotide” has a six-membered tetrahydropyran “sugar”substituted in for the pentofuranosyl residue in normal nucleotides (asugar surrogate). Modified THP nucleotides include, but are not limitedto, what is referred to in the art as hexitol nucleic acid (HNA), anitolnucleic acid (ANA), manitol nucleic acid (MNA) (see, e.g., Leumann,Bioorg. Med. Chem., 2002, 10, 841-854), or fluoro HNA (F-HNA).

In some embodiments of the invention, sugar surrogates comprise ringshaving more than 5 atoms and more than one heteroatom. For examplenucleotides comprising morpholino sugar moieties and their use inoligomeric compounds has been reported (see for example: Braasch et al.,Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685;5,166,315; 5,185,444; and 5,034,506). Morpholinos may be modified, forexample by adding or altering various substituent groups from the abovemorpholino structure. Such sugar surrogates are referred to herein as“modified morpholinos.”

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 published on Aug. 21, 2008 for otherdisclosed 5′, 2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH2-O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). The synthesis and preparation of carbocyclic bicyclicnucleosides along with their oligomerization and biochemical studieshave also been described (see, e.g., Srivastava et al., J. Am. Chem.Soc. 2007, 129(26), 8362-8379).

In certain embodiments, antisense compounds comprise one or moremodified cyclohexenyl nucleosides, which is a nucleoside having asix-membered cyclohexenyl in place of the pentofuranosyl residue innaturally occurring nucleosides. Modified cyclohexenyl nucleosidesinclude, but are not limited to those described in the art (see forexample commonly owned, published PCT Application WO 2010/036696,published on Apr. 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008,130(6), 1979-1984; Horvath et al., Tetrahedron Letters, 2007, 48,3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30),9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005,24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005,33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F:Structural Biology and Crystallization Communications, 2005, F61(6),585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al.,Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem.,2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001,29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wanget al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7),785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCTapplication, WO 06/047842; and Published PCT Application WO 01/049687;the text of each is incorporated by reference herein, in theirentirety).

An antisense polynucleotide agent can also include nucleobasemodifications or substitutions. As used herein, “unmodified” or“natural” nucleobases include the purine bases adenine (A) and guanine(G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified nucleobases include other synthetic and natural nucleobasessuch as deoxy-thymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl analother 8-substituted adenines and guanines, 5-halo, particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine.Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in “Modified Nucleosides in Biochemistry,” Biotechnologyand Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in TheConcise Encyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y S., Chapter 15, antisensepolynucleotide agent Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theagents featured in the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., Eds., antisense polynucleotide agent ResearchandApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and areexemplary base substitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

One or more of the nucleotides of an iRNA of the invention may alsoinclude a hydroxymethyl substituted nucleotide. A “hydroxymethylsubstituted nucleotide” is an acyclic 2′-3′-seco-nucleotide, alsoreferred to as an “unlocked nucleic acid” (“UNA”) modification.Representative U.S. publications that teach the preparation of UNAinclude, but are not limited to, U.S. Pat. No. 8,314,227; and US PatentPublication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, theentire contents of each of which are hereby incorporated herein byreference.

Additional modification which may potentially stabilize the ends ofantisense polynucleotide agents can includeN-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in US Patent PublicationNo. 2012/0142101.

Any of the antisense polynucleotide agents of the invention may beoptionally conjugated with a GalNAc derivative ligand, as described inSection IV, below.

As described in more detail below, an agent that contains conjugationsof one or more carbohydrate moieties to an antisense polynucleotideagent can optimize one or more properties of the agent. In many cases,the carbohydrate moiety will be attached to a modified subunit of theantisense polynucleotide agent. For example, the ribose sugar of one ormore ribonucleotide subunits of an agent can be replaced with anothermoiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which isattached a carbohydrate ligand. A ribonucleotide subunit in which theribose sugar of the subunit has been so replaced is referred to hereinas a ribose replacement modification subunit (RRMS). A cyclic carriermay be a carbocyclic ring system, i.e., all ring atoms are carbon atoms,or a heterocyclic ring system, i.e., one or more ring atoms may be aheteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be amonocyclic ring system, or may contain two or more rings, e.g. fusedrings. The cyclic carrier may be a fully saturated ring system, or itmay contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

The antisense polynucleotide agents may be conjugated to a ligand via acarrier, wherein the carrier can be cyclic group or acyclic group;preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl,pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl,[1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl,thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl,tetrahydrofuryl and and decalin; preferably, the acyclic group isselected from serinol backbone or diethanolamine backbone.

In certain specific embodiments, the antisense polynucleotide agent foruse in the methods of the invention is an agent selected from the groupof agents listed in Tables 3 and 4. These agents may further comprise aligand, as described in Section IV, below.

A. Polynucleotide Agents Comprising Motifs

In certain embodiments of the invention, at least one of the contiguousnucleotides of the polynucleotide agents of the invention, e.g., theantisense polynucleotide agents of the invention, may be a modifiednucleotide. In one embodiment, the modified nucleotide comprises one ormore modified sugars. In other embodiments, the modified nucleotidecomprises one or more modified nucleobases. In yet other embodiments,the modified nucleotide comprises one or more modified internucleosidelinkages. In some embodiments, the modifications (sugar modifications,nucleobase modifications, and/or linkage modifications) define a patternor motif. In one embodiment, the patterns of modifications of sugarmoieties, internucleoside linkages, and nucleobases are each independentof one another.

Antisense polynucleotide agents having modified oligonucleotidesarranged in patterns, or motifs may, for example, confer to the agentsproperties such as enhanced inhibitory activity, increased bindingaffinity for a target nucleic acid, or resistance to degradation by invivo nucleases. For example, such agents may contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, increased binding affinity for the targetnucleic acid, and/or increased inhibitory activity. A second region ofsuch agents may optionally serve as a substrate for the cellularendonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

An exemplary antisense polynucleotide agent having modifiedoligonucleotides arranged in patterns, or motifs is a gapmer. In a“gapmer”, an internal region or “gap” having a plurality of linkednucleotides that supports RNaseH cleavage is positioned between twoexternal flanking regions or “wings” having a plurality of linkednucleotides that are chemically distinct from the linked nucleotides ofthe internal region. The gap segment generally serves as the substratefor endonuclease cleavage, while the wing segments comprise modifiednucleotides.

The three regions of a gapmer motif (the 5′-wing, the gap, and the3′-wing) form a contiguous sequence of nucleotides and may be describedas “X-Y-Z”, wherein “X” represents the length of the 5-wing, “Y”represents the length of the gap, and “Z” represents the length of the3′-wing. In one embodiment, a gapmer described as “X-Y-Z” has aconfiguration such that the gap segment is positioned immediatelyadjacent to each of the 5′ wing segment and the 3′ wing segment. Thus,no intervening nucleotides exist between the 5′ wing segment and gapsegment, or the gap segment and the 3′ wing segment. Any of theantisense compounds described herein can have a gapmer motif. In someembodiments, X and Z are the same, in other embodiments they aredifferent.

In certain embodiments, the regions of a gapmer are differentiated bythe types of modified nucleotides in the region. The types of modifiednucleotides that may be used to differentiate the regions of a gapmer,in some embodiments, include β-D-ribonucleotides,β-D-deoxyribonucleotides, 2′-modified nucleotides, e.g., 2′-modifiednucleotides (e.g., 2′-MOE, and 2′-O—CH3), and bicyclic sugar modifiednucleotides (e.g., those having a 4′-(CH2)n-O-2′ bridge, where n=1 orn=2).

In one embodiment, at least some of the modified nucleotides of each ofthe wings may differ from at least some of the modified nucleotides ofthe gap. For example, at least some of the modified nucleotides of eachwing that are closest to the gap (the 3′-most nucleotide of the 5′-wingand the 5′-most nucleotide of the 3-wing) differ from the modifiednucleotides of the neighboring gap nucleotides, thus defining theboundary between the wings and the gap. In certain embodiments, themodified nucleotides within the gap are the same as one another. Incertain embodiments, the gap includes one or more modified nucleotidesthat differ from the modified nucleotides of one or more othernucleotides of the gap.

The length of the 5′-wing (X) of a gapmer may be 1 to 6 nucleotides inlength, e.g., 2 to 6, 2 to 5, 3 to 6, 3 to 5, 1 to 5, 1 to 4, 1 to 3, 2to 4 nucleotides in length, e.g., 1, 2, 3, 4, 5, or 6 nucleotides inlength.

The length of the 3′-wing (Z) of a gapmer may be 1 to 6 nucleotides inlength, e.g., 2 to 6, 2-5, 3 to 6, 3 to 5, 1 to 5, 1 to 4, 1 to 3, 2 to4 nucleotides in length, e.g., 1, 2, 3, 4, 5, or 6 nucleotides inlength.

The length of the gap (Y) of a gapmer may be 5 to 14 nucleotides inlength, e.g., 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 5 to 8, 5 to7, 5 to 6, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, 6 to 8,6 to 7, 7 to 14, 7 to 13, 7 to 12, 7 to ii, 7 to 10, 7 to 9, 7 to 8, 8to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 8 to 9, 9 to 14, 9 to 13, 9to 12, 9 to 1, 9 to 0, 10 to 14, 10 to 13, 10 to 12, 10 to 11, 11 to 14,11 to 13, 11 to 12, 12 to 14, 12 to 13, or 13 to 14 nucleotides inlength, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides inlength.

In some embodiments of the invention X consists of 2, 3, 4, 5 or 6nucleotides, Y consists of 7, 8, 9, 10, 11, or 12 nucleotides, and Zconsists of 2, 3, 4, 5 or 6 nucleotides. Such gapmers include (X-Y-Z)2-7-2, 2-7-3, 2-7-4, 2-7-5, 2-7-6, 3-7-2, 3-7-3, 3-7-4, 3-7-5, 3-7-6,4-7-3, 4-7-4, 4-7-5, 4-7-6, 5-7-3, 5-7-4, 5-7-5, 5-7-6, 6-7-3, 6-7-4,6-7-5, 6-7-6, 3-7-3, 3-7-4, 3-7-5, 3-7-6, 4-7-3, 4-7-4, 4-7-5, 4-7-6,5-7-3, 5-7-4, 5-7-5, 5-7-6, 6-7-3, 6-7-4, 6-7-5, 6-7-6, 2-8-2, 2-8-3,2-8-4, 2-8-5, 2-8-6, 3-8-2, 3-8-3, 3-8-4, 3-8-5, 3-8-6, 4-8-3, 4-8-4,4-8-5, 4-8-6, 5-8-3, 5-8-4, 5-8-5, 5-8-6, 6-8-3, 6-8-4, 6-8-5, 6-8-6,2-9-2, 2-9-3, 2-9-4, 2-9-5, 2-9-6, 3-9-2, 3-9-3, 3-9-4, 3-9-5, 3-9-6,4-9-3, 4-9-4, 4-9-5, 4-9-6, 5-9-3, 5-9-4, 5-9-5, 5-9-6, 6-9-3, 6-9-4,6-9-5, 6-9-6, 2-10-2, 2-10-3, 2-10-4, 2-10-5, 2-10-6, 3-10-2, 3-10-3,3-10-4, 3-10-5, 3-10-6, 4-10-3, 4-10-4, 4-10-5, 4-10-6, 5-10-3, 5-10-4,5-10-5, 5-10-6, 6-10-3, 6-10-4, 6-10-5, 6-10-6, 2-11-2, 2-11-3, 2-11-4,2-11-5, 2-11-6, 3-11-2, 3-11-3, 3-11-4, 3-11-5, 3-11-6, 4-11-3, 4-11-4,4-11-5, 4-11-6, 5-11-3, 5-11-4, 5-11-5, 5-11-6, 6-11-3, 6-11-4, 6-11-5,6-11-6, 2- 12-2, 2-12-3, 2-12-4, 2-12-5, 2-12-6, 3-12-2, 3-12-3, 3-12-4,3-12-5, 3-12-6, 4-12-3, 4-12-4, 4-12-5, 4-12-6, 5-12-3, 5-12-4, 5-12-5,5-12-6, 6-12-3, 6-12-4, 6-12-5, or 6-12-6.

In some embodiments of the invention, antisense polynucleotide agentstargeting ALAS1 include a 5-10-5 gapmer motif. In some embodiments ofthe invention, antisense polynucleotide agents targeting ALAS1 include a5-11-5 gapmer motif. In other embodiments of the invention, antisensepolynucleotide agents targeting ALAS1 include a 4-10-4 gapmer motif. Inother embodiments of the invention, antisense polynucleotide agentstargeting ALAS1 include a 4-11-4 gapmer motif. In another embodiment ofthe invention, antisense polynucleotide agents targeting ALAS1 include a3-10-3 gapmer motif. In other embodiments of the invention, antisensepolynucleotide agents targeting ALAS1 include a 3-11-3 gapmer motif. Inyet other embodiments of the invention, antisense polynucleotide agentstargeting ALAS1 include a 2-10-2 gapmer motif. In other embodiments ofthe invention, antisense polynucleotide agents targeting ALAS1 include a2-11-2 gapmer motif.

The 5′-wing and/or 3′-wing of a gapmer may independently include 1-6modified nucleotides, e.g., 1, 2, 3, 4, 5, or 6 modified nucleotides.

In some embodiment, the 5′-wing of a gapmer includes at least onemodified nucleotide. In one embodiment, the 5′-wing of a gapmercomprises at least two modified nucleotides. In another embodiment, the5′-wing of a gapmer comprises at least three modified nucleotides. Inyet another embodiment, the 5′-wing of a gapmer comprises at least fourmodified nucleotides. In another embodiment, the 5′-wing of a gapmercomprises at least five modified nucleotides. In certain embodiments,each nucleotide of the 5′-wing of a gapmer is a modified nucleotide.

In some embodiments, the 3′-wing of a gapmer includes at least onemodified nucleotide. In one embodiment, the 3′-wing of a gapmercomprises at least two modified nucleotides. In another embodiment, the3′-wing of a gapmer comprises at least three modified nucleotides. Inyet another embodiment, the 3′-wing of a gapmer comprises at least fourmodified nucleotides. In another embodiment, the 3′-wing of a gapmercomprises at least five modified nucleotides. In certain embodiments,each nucleotide of the 3′-wing of a gapmer is a modified nucleotide.

In certain embodiments, the regions of a gapmer are differentiated bythe types of sugar moieties of the nucleotides. In one embodiment, thenucleotides of each distinct region comprise uniform sugar moieties. Inother embodiments, the nucleotides of each distinct region comprisedifferent sugar moieties. In certain embodiments, the sugar nucleotidemodification motifs of the two wings are the same as one another. Incertain embodiments, the sugar nucleotide modification motifs of the5′-wing differs from the sugar nucleotide modification motif of the3′-wing.

The 5′-wing of a gapmer may include 1-6 modified nucleotides, e.g., 1,2, 3, 4, 5, or 6 modified nucleotides.

In one embodiment, at least one modified nucleotide of the 5′-wing of agapmer is a bicyclic nucleotide, such as a constrained ethyl nucleotide,or an LNA. In another embodiment, the 5′-wing of a gapmer includes 2, 3,4, or 5 bicyclic nucleotides. In some embodiments, each nucleotide ofthe 5′-wing of a gapmer is a bicyclic nucleotide.

In one embodiment, the 5′-wing of a gapmer includes at least 1, 2, 3, 4,or 5 constrained ethyl nucleotides. In some embodiments, each nucleotideof the 5′-wing of a gapmer is a constrained ethyl nucleotide.

In one embodiment, the 5′-wing of a gapmer comprises at least one LNAnucleotide. In another embodiment, the 5′-wing of a gapmer includes 2,3, 4, or 5 LNA nucleotides. In other embodiments, each nucleotide of the5′-wing of a gapmer is an LNA nucleotide.

In certain embodiments, at least one modified nucleotide of the 5′-wingof a gapmer is a non-bicyclic modified nucleotide, e.g., a2′-substituted nucleotide. A “2′-substituted nucleotide” is a nucleotidecomprising a modification at the 2′-position which is other than H orOH, such as a 2′-OMe nucleotide, or a 2′-MOE nucleotide. In oneembodiment, the 5′-wing of a gapmer comprises 2, 3, 4, or 52′-substituted nucleotides. In one embodiment, each nucleotide of the5′-wing of a gapmer is a 2′-substituted nucleotide.

In one embodiment, the 5′-wing of a gapmer comprises at least one 2′-OMenucleotide. In one embodiment, the 5′-wing of a gapmer comprises atleast 2, 3, 4, or 5 2′-OMe nucleotides. In one embodiment, each of thenucleotides of the 5′-wing of a gapmer comprises a 2′-OMe nucleotide.

In one embodiment, the 5′-wing of a gapmer comprises at least one 2′-MOEnucleotide. In one embodiment, the 5′-wing of a gapmer comprises atleast 2, 3, 4, or 5 2′-MOE nucleotides. In one embodiment, each of thenucleotides of the 5′-wing of a gapmer comprises a 2′-MOE nucleotide.

In certain embodiments, the 5′-wing of a gapmer comprises at least one2′-deoxynucleotide. In certain embodiments, each nucleotide of the5′-wing of a gapmer is a 2′-deoxynucleotide. In a certain embodiments,the 5′-wing of a gapmer comprises at least one ribonucleotide. Incertain embodiments, each nucleotide of the 5′-wing of a gapmer is aribonucleotide.

The 3′-wing of a gapmer may include 1-6 modified nucleotides, e.g., 1,2, 3, 4, 5, or 6 modified nucleotides.

In one embodiment, at least one modified nucleotide of the 3′-wing of agapmer is a bicyclic nucleotide, such as a constrained ethyl nucleotide,or an LNA. In another embodiment, the 3′-wing of a gapmer includes 2, 3,4, or 5 bicyclic nucleotides. In some embodiments, each nucleotide ofthe 3′-wing of a gapmer is a bicyclic nucleotide.

In one embodiment, the 3′-wing of a gapmer includes at least oneconstrained ethyl nucleotide. In another embodiment, the 3′-wing of agapmer includes 2, 3, 4, or 5 constrained ethyl nucleotides. In someembodiments, each nucleotide of the 3′-wing of a gapmer is a constrainedethyl nucleotide.

In one embodiment, the 3′-wing of a gapmer comprises at least one LNAnucleotide. In another embodiment, the 3′-wing of a gapmer includes 2,3, 4, or 5 LNA nucleotides. In other embodiments, each nucleotide of the3′-wing of a gapmer is an LNA nucleotide.

In certain embodiments, at least one modified nucleotide of the 3′-wingof a gapmer is a non-bicyclic modified nucleotide, e.g., a2′-substituted nucleotide. In one embodiment, the 3′-wing of a gapmercomprises 2, 3, 4, or 5 2′-substituted nucleotides. In one embodiment,each nucleotide of the 3′-wing of a gapmer is a 2′-substitutednucleotide.

In one embodiment, the 3′-wing of a gapmer comprises at least one 2′-OMenucleotide. In one embodiment, the 3′-wing of a gapmer comprises atleast 2, 3, 4, or 5 2′-OMe nucleotides. In one embodiment, each of thenucleotides of the 3′-wing of a gapmer comprises a 2′-OMe nucleotide.

In one embodiment, the 3′-wing of a gapmer comprises at least one 2′-MOEnucleotide. In one embodiment, the 3′-wing of a gapmer comprises atleast 2, 3, 4, or 5 2′-MOE nucleotides. In one embodiment, each of thenucleotides of the 3′-wing of a gapmer comprises a 2′-MOE nucleotide.

In certain embodiments, the 3′-wing of a gapmer comprises at least one2′-deoxynucleotide. In certain embodiments, each nucleotide of the3′-wing of a gapmer is a 2′-deoxynucleotide. In a certain embodiments,the 3′-wing of a gapmer comprises at least one ribonucleotide. Incertain embodiments, each nucleotide of the 3′-wing of a gapmer is aribonucleotide.

The gap of a gapmer may include 5-14 modified nucleotides, e.g., 5, 6,7, 8, 9, 10, 11, 12, 13, or 14 modified nucleotides.

In one embodiment, the gap of a gapmer comprises at least one5-methylcytosine. In one embodiment, the gap of a gapmer comprises atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 5-methylcytosines. Inone embodiment, all of the nucleotides of the the gap of a gapmer are5-methylcytosines.

In one embodiment, the gap of a gapmer comprises at least one2′-deoxynucleotide. In one embodiment, the gap of a gapmer comprises atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 2′-deoxynucleotides. Inone embodiment, all of the nucleotides of the the gap of a gapmer are2′-deoxynucleotides.

A gapmer may include one or more modified internucleotide linkages. Insome embodiments, a gapmer includes one or more phosphodiesterinternucleotide linkages. In other embodiments, a gapmer includes one ormore phosphorothioate internucleotide linkages.

In one embodiment, each nucleotide of a 5′-wing of a gapmer are linkedvia a phosphorothioate internucleotide linkage. In another embodiment,each nucleotide of a 3′-wing of a gapmer are linked via aphosphorothioate internucleotide linkage. In yet another embodiment,each nucleotide of a gap segment of a gapmer is linked via aphosphorothioate internucleotide linkage. In one embodiment, all of thenucleotides in a gapmer are linked via phosphorothioate internucleotidelinkages.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fivenucleotides and a 3′-wing segment comprising 5 nucleotides.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising five nucleotides and a 3′-wing segment comprising 5nucleotides.

In another embodiment, an antisense polynucleotide agent targeting anALAS1 gene comprises a gap segment of ten 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising four nucleotides and a 3′-wing segment comprising fournucleotides.

In another embodiment, an antisense polynucleotide agent targeting anALAS1 gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising four nucleotides and a 3′-wing segment comprising fournucleotides.

In another embodiment, an antisense polynucleotide agent targeting anALAS1 gene comprises a gap segment of ten 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising three nucleotides and a 3′-wing segment comprising threenucleotides.

In another embodiment, an antisense polynucleotide agent targeting anALAS1 gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising three nucleotides and a 3′-wing segment comprising threenucleotides.

In another embodiment, an antisense polynucleotide agent targeting anALAS1 gene comprises a gap segment of ten 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising two nucleotides and a 3′-wing segment comprising twonucleotides.

In another embodiment, an antisense polynucleotide agent targeting anALAS1 gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising two nucleotides and a 3′-wing segment comprising twonucleotides.

In one embodiment, each nucleotide of a 5-wing flanking a gap segment of10 2′-deoxyribonucleotides comprises a modified nucleotide. In anotherembodiment, each nucleotide of a 3-wing flanking a gap segment of 102′-deoxyribonucleotides comprises a modified nucleotide. In anotherembodiment, each nucleotide of a 3-wing flanking a gap segment of 112′-deoxyribonucleotides comprises a modified nucleotide. In oneembodiment, each of the modified 5′-wing nucleotides and each of themodified 3′-wing nucleotides comprise a 2′-sugar modification. In oneembodiment, the 2′-sugar modification is a 2′-OMe modification. Inanother embodiment, the 2′-sugar modification is a 2′-MOE modification.In one embodiment, each of the modified 5′-wing nucleotides and each ofthe modified 3′-wing nucleotides comprise a bicyclic nucleotide. In oneembodiment, the bicyclic nucleotide is a constrained ethyl nucleotide.In another embodiment, the bicyclic nucleotide is an LNA nucleotide. Inone embodiment, each cytosine in an antisense polynucleotide agenttargeting an ALAS1 gene is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fivenucleotides comprising a 2′OMe modification and a 3′-wing segmentcomprising five nucleotides comprising a 2′OMe modification, whereineach internucleotide linkage of the agent is a phosphorothioate linkage.In one embodiment, each cytosine of the agent is a 5-methylcytosine. Inone embodiment, the agent further comprises a ligand.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising five nucleotides comprising a 2′OMe modification and a3′-wing segment comprising five nucleotides comprising a 2′OMemodification, wherein each internucleotide linkage of the agent is aphosphorothioate linkage. In one embodiment, each cytosine of the agentis a 5-methylcytosine. In one embodiment, the agent further comprises aligand.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fivenucleotides comprising a 2′MOE modification and a 3′-wing segmentcomprising five nucleotides comprising a 2′MOE modification, whereineach internucleotide linkage of the agent is a phosphorothioate linkage.In one embodiment, each cytosine of the agent is a 5-methylcytosine. Inone embodiment, the agent further comprises a ligand.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising five nucleotides comprising a 2′MOE modification and a3′-wing segment comprising five nucleotides comprising a 2′MOEmodification, wherein each internucleotide linkage of the agent is aphosphorothioate linkage. In one embodiment, each cytosine of the agentis a 5-methylcytosine. In one embodiment, the agent further comprises aligand.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fiveconstrained ethyl nucleotides and a 3′-wing segment comprising fiveconstrained ethyl nucleotides, wherein each internucleotide linkage ofthe agent is a phosphorothioate linkage. In one embodiment, eachcytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising five constrained ethyl nucleotides and a 3′-wing segmentcomprising five constrained ethyl nucleotides, wherein eachinternucleotide linkage of the agent is a phosphorothioate linkage. Inone embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fiveLNA nucleotides and a 3′-wing segment comprising five LNA nucleotides,wherein each internucleotide linkage of the agent is a phosphorothioatelinkage. In one embodiment, each cytosine of the agent is a5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising five LNA nucleotides and a 3′-wing segment comprising fiveLNA nucleotides, wherein each internucleotide linkage of the agent is aphosphorothioate linkage. In one embodiment, each cytosine of the agentis a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fournucleotides comprising a 2′OMe modification and a 3′-wing segmentcomprising four nucleotides comprising a 2′OMe modification, whereineach internucleotide linkage of the agent is a phosphorothioate linkage.In one embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising four nucleotides comprising a 2′OMe modification and a3′-wing segment comprising four nucleotides comprising a 2′OMemodification, wherein each internucleotide linkage of the agent is aphosphorothioate linkage. In one embodiment, each cytosine of the agentis a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fournucleotides comprising a 2′MOE modification and a 3′-wing segmentcomprising four nucleotides comprising a 2′MOE modification, whereineach internucleotide linkage of the agent is a phosphorothioate linkage.In one embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising four nucleotides comprising a 2′MOE modification and a3′-wing segment comprising four nucleotides comprising a 2′MOEmodification, wherein each internucleotide linkage of the agent is aphosphorothioate linkage. In one embodiment, each cytosine of the agentis a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fourconstrained ethyl nucleotides and a 3′-wing segment comprising fourconstrained ethyl nucleotides, wherein each internucleotide linkage ofthe agent is a phosphorothioate linkage. In one embodiment, eachcytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising four constrained ethyl nucleotides and a 3′-wing segmentcomprising four constrained ethyl nucleotides, wherein eachinternucleotide linkage of the agent is a phosphorothioate linkage. Inone embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fourLNA nucleotides and a 3′-wing segment comprising four LNA nucleotides,wherein each internucleotide linkage of the agent is a phosphorothioatelinkage. In one embodiment, each cytosine of the agent is a5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising four LNA nucleotides and a 3′-wing segment comprising fourLNA nucleotides, wherein each internucleotide linkage of the agent is aphosphorothioate linkage. In one embodiment, each cytosine of the agentis a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising threenucleotides comprising a 2′OMe modification and a 3′-wing segmentcomprising three nucleotides comprising a 2′OMe modification, whereineach internucleotide linkage of the agent is a phosphorothioate linkage.In one embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising three nucleotides comprising a 2′OMe modification and a3′-wing segment comprising three nucleotides comprising a 2′OMemodification, wherein each internucleotide linkage of the agent is aphosphorothioate linkage. In one embodiment, each cytosine of the agentis a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising threenucleotides comprising a 2′MOE modification and a 3′-wing segmentcomprising three nucleotides comprising a 2′MOE modification, whereineach internucleotide linkage of the agent is a phosphorothioate linkage.In one embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising three nucleotides comprising a 2′MOE modification and a3′-wing segment comprising three nucleotides comprising a 2′MOEmodification, wherein each internucleotide linkage of the agent is aphosphorothioate linkage. In one embodiment, each cytosine of the agentis a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising threeconstrained ethyl nucleotides and a 3′-wing segment comprising threeconstrained ethyl nucleotides, wherein each internucleotide linkage ofthe agent is a phosphorothioate linkage. In one embodiment, eachcytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising three constrained ethyl nucleotides and a 3′-wing segmentcomprising three constrained ethyl nucleotides, wherein eachinternucleotide linkage of the agent is a phosphorothioate linkage. Inone embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting a anALAS1 gene comprises a gap segment of ten 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising three LNA nucleotides and a 3′-wing segment comprising threeLNA nucleotides, wherein each internucleotide linkage of the agent is aphosphorothioate linkage. In one embodiment, each cytosine of the agentis a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting a anALAS1 gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising three LNA nucleotides and a 3′-wing segment comprising threeLNA nucleotides, wherein each internucleotide linkage of the agent is aphosphorothioate linkage. In one embodiment, each cytosine of the agentis a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising twonucleotides comprising a 2′OMe modification and a 3′-wing segmentcomprising two nucleotides comprising a 2′OMe modification, wherein eachinternucleotide linkage of the agent is a phosphorothioate linkage. Inone embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising two nucleotides comprising a 2′OMe modification and a 3′-wingsegment comprising two nucleotides comprising a 2′OMe modification,wherein each internucleotide linkage of the agent is a phosphorothioatelinkage. In one embodiment, each cytosine of the agent is a5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising twonucleotides comprising a 2′MOE modification and a 3′-wing segmentcomprising two nucleotides comprising a 2′MOE modification, wherein eachinternucleotide linkage of the agent is a phosphorothioate linkage. Inone embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising two nucleotides comprising a 2′MOE modification and a 3′-wingsegment comprising two nucleotides comprising a 2′MOE modification,wherein each internucleotide linkage of the agent is a phosphorothioatelinkage. In one embodiment, each cytosine of the agent is a5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising twoconstrained ethyl nucleotides and a 3′-wing segment comprising twoconstrained ethyl nucleotides, wherein each internucleotide linkage ofthe agent is a phosphorothioate linkage. In one embodiment, eachcytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising two constrained ethyl nucleotides and a 3′-wing segmentcomprising two constrained ethyl nucleotides, wherein eachinternucleotide linkage of the agent is a phosphorothioate linkage. Inone embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising two LNAnucleotides and a 3′-wing segment comprising two LNA nucleotides,wherein each internucleotide linkage of the agent is a phosphorothioatelinkage. In one embodiment, each cytosine of the agent is a5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting an ALAS1gene comprises a gap segment of eleven 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising two LNA nucleotides and a 3′-wing segment comprising two LNAnucleotides, wherein each internucleotide linkage of the agent is aphosphorothioate linkage. In one embodiment, each cytosine of the agentis a 5-methylcytosine.

Further gapmer designs suitable for use in the agents, compositions, andmethods of the invention are disclosed in, for example, U.S. Pat. Nos.7,687,617 and 8,580,756; U.S. Patent Publication Nos. 20060128646,20090209748, 20140128586, 20140128591, 20100210712, and 20080015162A1;and International Publication No. WO 2013/159108, the entire content ofeach of which are incorporated herein by reference.

IV. Polynucleotide Agents Conjugated to Ligands

Another modification of the polynucleotide agents of the inventioninvolves chemically linking to the agent one or more ligands, moietiesor conjugates that enhance the activity, cellular distribution orcellular uptake of the antisense polynucleotide agent. Such moietiesinclude but are not limited to lipid moieties such as a cholesterolmoiety (Letsinger et al., Proc. Natl Acid. Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994,4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al.,Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med.Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuket al., Biochimie, 1993, 75:49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res.,1990, 18:3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of an antisense polynucleotide agent into which it isincorporated. In preferred embodiments a ligand provides an enhancedaffinity for a selected target, e.g., molecule, cell or cell type,compartment, e.g., a cellular or organ compartment, tissue, organ orregion of the body, as, e.g., compared to a species absent such aligand. Preferred ligands will not take part in hybridization of anantisense polynucleotide agent to the targeted mRNA.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or alipid. The ligand can also be a recombinant or synthetic molecule, suchas a synthetic polymer, e.g., a synthetic polyamino acid. Examples ofpolyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucoseamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGDpeptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a hepaticcell. Ligands can also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalentmannose, or multivalent fucose. The ligand can be, for example, alipopolysaccharide, an activator of p38 MAP kinase, or an activator ofNF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the antisense polynucleotide agent into the cell, for example,by disrupting the cell's cytoskeleton, e.g., by disrupting the cell'smicrotubules, microfilaments, and/or intermediate filaments. The drugcan be, for example, taxon, vincristine, vinblastine, cytochalasin,nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A,indanocine, or myoservin.

In some embodiments, a ligand attached to an antisense polynucleotideagent as described herein acts as a pharmacokinetic modulator (PKmodulator). PK modulators include lipophiles, bile acids, steroids,phospholipid analogues, peptides, protein binding agents, PEG, vitaminsetc. Exemplary PK modulators include, but are not limited to,cholesterol, fatty acids, cholic acid, lithocholic acid,dialkylglycerides, diacylglyceride, phospholipids, sphingolipids,naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides thatcomprise a number of phosphorothioate linkages are also known to bind toserum protein, thus short oligonucleotides, e.g., oligonucleotides ofabout 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple ofphosphorothioate linkages in the backbone are also amenable to thepresent invention as ligands (e.g. as PK modulating ligands). Inaddition, aptamers that bind serum components (e.g. serum proteins) arealso suitable for use as PK modulating ligands in the embodimentsdescribed herein.

Ligand-conjugated polynucleotides of the invention may be synthesized bythe use of a polynucleotide that bears a pendant reactive functionality,such as that derived from the attachment of a linking molecule onto theoligonucleotide (described below). This reactive polynucleotide may bereacted directly with commercially-available ligands, ligands that aresynthesized bearing any of a variety of protecting groups, or ligandsthat have a linking moiety attached thereto.

The polynucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other polynucleotides, such as thephosphorothioates and alkylated derivatives.

In the ligand-conjugated polynucleotides and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, thepolynucleotides and polynucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide. In someembodiments, the polynucleotides or linked nucleosides of the presentinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

A. Lipid Conjugates

In one embodiment, the ligand or conjugate is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, naproxen oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bytarget cells such as liver cells. Also included are HSA and low densitylipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to antisensepolynucleotide agents can affect pharmacokinetic distribution of theagent, such as by enhancing cellular recognition and absorption. Thepeptide or peptidomimetic moiety can be about 5-50 amino acids long,e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 3). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 4) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 5) and theDrosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 6) havebeen found to be capable of functioning as delivery peptides. A peptideor peptidomimetic can be encoded by a random sequence of DNA, such as apeptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to anantisens epolynucleotide agent via an incorporated monomer unit for celltargeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide,or RGD mimic. A peptide moiety can range in length from about 5 aminoacids to about 40 amino acids. The peptide moieties can have astructural modification, such as to increase stability or directconformational properties. Any of the structural modifications describedbelow can be utilized.

An RGD peptide for use in the compositions and methods of the inventionmay be linear or cyclic, and may be modified, e.g., glycosylated ormethylated, to facilitate targeting to a specific tissue(s).RGD-containing peptides and peptidiomimemtics may include D-amino acids,as well as synthetic RGD mimics. In addition to RGD, one can use othermoieties that target the integrin ligand. Preferred conjugates of thisligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, anantisense polynucleotide agent further comprises a carbohydrate. Thecarbohydrate conjugated agents are advantageous for the in vivo deliveryof nucleic acids, as well as compositions suitable for in vivotherapeutic use, as described herein (see, e.g., Prakash, et al. (2014)Nuc Acid Res doi 10.1093/nar/gku531). As used herein, “carbohydrate”refers to a compound which is either a carbohydrate per se made up ofone or more monosaccharide units having at least 6 carbon atoms (whichcan be linear, branched or cyclic) with an oxygen, nitrogen or sulfuratom bonded to each carbon atom; or a compound having as a part thereofa carbohydrate moiety made up of one or more monosaccharide units eachhaving at least six carbon atoms (which can be linear, branched orcyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbonatom. Representative carbohydrates include the sugars (mono-, di-, tri-and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include C5and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharidesinclude sugars having two or three monosaccharide units (e.g., C5, C6,C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositionsand methods of the invention is a monosaccharide. In one embodiment, themonosaccharide is an N-acetylgalactosamine, such as

In another embodiment, a carbohydrate conjugate for use in thecompositions and methods of the invention is selected from the groupconsisting of:

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator and/or a cell permeation peptide.

D. Linkers

In some embodiments, the conjugate or ligand described herein can beattached to an antisense polynucleotide agent with various linkers thatcan be cleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between about1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17,8-17, 6-16, 7-16, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least about 10 times, 20,times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times or more, or at least about 100 times faster in a target cell orunder a first reference condition (which can, e.g., be selected to mimicor represent intracellular conditions) than in the blood of a subject,or under a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, orabout 100 times faster in the cell (or under in vitro conditionsselected to mimic intracellular conditions) as compared to blood orserum (or under in vitro conditions selected to mimic extracellularconditions).

i. Redox cleavable linking groups

In one embodiment, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(—S—S—). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular antisense polynucleotide agent moietyand particular targeting agent one can look to methods described herein.For example, a candidate can be evaluated by incubation withdithiothreitol (DTT), or other reducing agent using reagents know in theart, which mimic the rate of cleavage which would be observed in a cell,e.g., a target cell. The candidates can also be evaluated underconditions which are selected to mimic blood or serum conditions. Inone, candidate compounds are cleaved by at most about 10% in the blood.In other embodiments, useful candidate compounds are degraded at leastabout 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 timesfaster in the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood (or under in vitroconditions selected to mimic extracellular conditions). The rate ofcleavage of candidate compounds can be determined using standard enzymekinetics assays under conditions chosen to mimic intracellular media andcompared to conditions chosen to mimic extracellular media.

ii. Phosphate-based cleavable linking groups

In another embodiment, a cleavable linker comprises a phosphate-basedcleavable linking group. A phosphate-based cleavable linking group iscleaved by agents that degrade or hydrolyze the phosphate group. Anexample of an agent that cleaves phosphate groups in cells are enzymessuch as phosphatases in cells. Examples of phosphate-based linkinggroups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodimentsare —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—,—O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—,—O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—,—S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—.These candidates can be evaluated using methods analogous to thosedescribed above.

iii. Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower),or by agents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

iv. Ester-Based Linking Groups

In another embodiment, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include but are not limited toesters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

v. Peptide-Based Cleaving Groups

In yet another embodiment, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

In one embodiment, an antisense polynucleotide agent of the invention isconjugated to a carbohydrate through a linker. Non-limiting examples ofantisense polynucleotide agent carbohydrate conjugates with linkers ofthe compositions and methods of the invention include, but are notlimited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention,a ligand is one or more “GalNAC” (N-acetylgalactosamine) derivativesattached through a bivalent or trivalent branched linker.

In one embodiment, a antisense polynucleotide agent of the invention isconjugated to a bivalent or trivalent branched linker selected from thegroup of structures shown in any of formula (XXXII)-(XXXV):

wherein:q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentlyfor each occurrence 0-20 and wherein the repeating unit can be the sameor different;P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherein one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H oramino acid side chain. Trivalent conjugating GalNAc derivatives areparticularly useful for use with antisense polynucleotide agents forinhibiting the expression of a target gene, such as those of formula(XXXVI):

-   -   wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide,        such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thestructures recited above as formulas II, VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; 8,106,022, the entire contents of each of whichare hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an antisense polynucleotide agent. The presentinvention also includes antisense polynucleotide agents that arechimeric compounds.

“Chimeric” antisense polynucleotide agents or “chimeras,” in the contextof this invention, are antisense polynucleotide agent compounds, whichcontain two or more chemically distinct regions, each made up of atleast one monomer unit, i.e., a nucleotide in the case of an antisensepolynucleotide agent. These antisense polynucleotide agents typicallycontain at least one region wherein the RNA is modified so as to conferupon the antisense polynucleotide agent increased resistance to nucleasedegradation, increased cellular uptake, and/or increased bindingaffinity for the target nucleic acid. An additional region of theantisense polynucleotide agent can serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency of antisensepolynucleotide agent inhibition of gene expression. Consequently,comparable results can often be obtained with shorter antisensepolynucleotide agents when chimeric antisense polynucleotide agents areused, compared to phosphorothioate deoxy antisense polynucleotide agentshybridizing to the same target region. Cleavage of the RNA target can beroutinely detected by gel electrophoresis and, if necessary, associatednucleic acid hybridization techniques known in the art.

In certain instances, the nucleotide of an antisense polynucleotideagent can be modified by a non-ligand group. A number of non-ligandmolecules have been conjugated to antisense polynucleotide agents inorder to enhance the activity, cellular distribution or cellular uptakeof the antisense polynucleotide agent, and procedures for performingsuch conjugations are available in the scientific literature. Suchnon-ligand moieties have included lipid moieties, such as cholesterol(Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61;Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholicacid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993,3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992,20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBSLett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), aphospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof an RNAs bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction can be performed either with the RNA still bound tothe solid support or following cleavage of the RNA, in solution phase.Purification of the RNA conjugate by HPLC typically affords the pureconjugate.

V. Delivery of a Polynucleotide Agent of the Invention

The delivery of a polynucleotide agent of the invention, e.g., anantisense polynucleotide agent of the invention, to a cell e.g., a cellwithin a subject, such as a human subject (e.g., a subject in needthereof, such as a subject having an ALAS1-associated disease) can beachieved in a number of different ways. For example, delivery may beperformed by contacting a cell with an antisense polynucleotide agent ofthe invention either in vitro or in vivo. In vivo delivery may also beperformed directly by administering a composition comprising anantisense polynucleotide agent to a subject.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with an antisense polynucleotideagent of the invention (see e.g., Akhtar S. and Julian R L. (1992)Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporatedherein by reference in their entireties). For in vivo delivery, factorsto consider in order to deliver an antisense polynucleotide agentinclude, for example, biological stability of the delivered molecule,prevention of non-specific effects, and accumulation of the deliveredmolecule in the target tissue. The non-specific effects of an antisensepolynucleotide agent can be minimized by local administration, forexample, by direct injection or implantation into a tissue or topicallyadministering the preparation. Local administration to a treatment sitemaximizes local concentration of the agent, limits the exposure of theagent to systemic tissues that can otherwise be harmed by the agent orthat can degrade the agent, and permits a lower total dose of theantisense polynucleotide agent to be administered. Several studies haveshown successful knockdown of gene products when an antisensepolynucleotide agent is administered locally. For example, intraoculardelivery of a VEGF antisense polynucleotide agent by intravitrealinjection in cynomolgus monkeys (Tolentino, M J., et al (2004) Retina24:132-138) and subretinal injections in mice (Reich, S J., et al (2003)Mol. Vis. 9:210-216) were both shown to prevent neovascularization in anexperimental model of age-related macular degeneration. In addition,direct intratumoral injection of a antisense polynucleotide agent inmice reduces tumor volume (Pille, J., et al (2005)Mol. Ther. 11:267-274)and can prolong survival of tumor-bearing mice (Kim, W J., et al (2006)Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNAinterference has also shown success with local delivery to the CNS bydirect injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMCNeurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528;Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering an antisense polynucleotide agent systemically for thetreatment of a disease, the agent can be modified or alternativelydelivered using a drug delivery system; both methods act to prevent therapid degradation of the antisense polynucleotide agent by endo- andexo-nucleases in vivo. Modification of the agent or the pharmaceuticalcarrier can also permit targeting of the antisense polynucleotide agentcomposition to the target tissue and avoid undesirable off-targeteffects. Antisense polynucleotide agent can be modified by chemicalconjugation to lipophilic groups such as cholesterol to enhance cellularuptake and prevent degradation. In an alternative embodiment, theantisense polynucleotide agent can be delivered using drug deliverysystems such as a nanoparticle, a dendrimer, a polymer, liposomes, or acationic delivery system. Positively charged cationic delivery systemsfacilitate binding of an antisense polynucleotide agent molecule(negatively charged) and also enhance interactions at the negativelycharged cell membrane to permit efficient uptake of an antisensepolynucleotide agent by the cell. Cationic lipids, dendrimers, orpolymers can either be bound to an antisense polynucleotide agent, orinduced to form a vesicle or micelle (see e.g., Kim S H., et al (2008)Journal of Controlled Release 129(2):107-116) that encases an antisensepolynucleotide agent. The formation of vesicles or micelles furtherprevents degradation of the antisense polynucleotide agent whenadministered systemically. Methods for making and administeringcationic-antisense polynucleotide agent complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et al(2003) J. Mol. Biol 327:761-766; Verma, U N, et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of antisense polynucleotide agents include DOTAP (Sorensen, DR., et al (2003), supra; Verma, U N., et al (2003), supra),Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S.,et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y., et al (2005)Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol.26:1087-1091), polyethyleneimine (Bonnet M E., et al (2008) Pharm. Res.August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol.71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm.3:472-487), and polyamidoamines (Tomalia, D A., et al (2007) Biochem.Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804).In some embodiments, an antisense polynucleotide agent forms a complexwith cyclodextrin for systemic administration. Methods foradministration and pharmaceutical compositions of antisensepolynucleotide agents and cyclodextrins can be found in U.S. Pat. No.7,427,605, which is herein incorporated by reference in its entirety.

VI. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions andformulations which include the polynucleotide agents of the invention.In one embodiment, provided herein are pharmaceutical compositionscontaining an antisense polynucleotide agent, as described herein, and apharmaceutically acceptable carrier.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human subjects and animal subjects without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the subject being treated. Some examples ofmaterials which can serve as pharmaceutically-acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)lubricating agents, such as magnesium state, sodium lauryl sulfate andtalc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pHbuffered solutions; (21) polyesters, polycarbonates and/orpolyanhydrides; (22) bulking agents, such as polypeptides and aminoacids (23) serum components, such as serum albumin, HDL and LDL; and(22) other non-toxic compatible substances employed in pharmaceuticalformulations.

The pharmaceutical compositions containing the antisense polynucleotideagents are useful for treating a disease or disorder associated with theexpression or activity of an ALAS1 gene, e.g. an ALAS1-associateddisease. Such pharmaceutical compositions are formulated based on themode of delivery. One example is compositions that are formulated forsystemic administration via parenteral delivery, e.g., by subcutaneous(SC) or intravenous (IV) delivery. Another example is compositions thatare formulated for direct delivery into the brain parenchyma, e.g., byinfusion into the brain, such as by continuous pump infusion. Thepharmaceutical compositions of the invention may be administered indosages sufficient to inhibit expression of an ALAS1 gene. In general, asuitable dose of an antisense polynucleotide agent of the invention willbe in the range of about 0.001 to about 200.0 milligrams per kilogrambody weight of the recipient per day, generally in the range of about 1to 50 mg per kilogram body weight per day. For example, the antisensepolynucleotide agent can be administered at about 0.01 mg/kg, about 0.05mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg,about 3 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40mg/kg, or about 50 mg/kg per single dose.

For example, the antisense polynucleotide agent may be administered at adose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 2, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, or about 50 mg/kg. Values and ranges intermediate to the recitedvalues are also intended to be part of this invention.

In another embodiment, the antisense polynucleotide agent isadministered at a dose of about 0.1 to about 50 mg/kg, about 0.25 toabout 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 to about 50mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35to about 50 mg/kg, about to about 50 mg/kg, about 45 to about 50 mg/kg,about 0.1 to about 45 mg/kg, about 0.25 to about 45 mg/kg, about 0.5 toabout 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg,about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 toabout 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg,about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about45 mg/kg, about 7.5 to about 45 mg/kg, about to about 45 mg/kg, about 15to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45mg/kg, about 0.1 to about 40 mg/kg, about 0.25 to about 40 mg/kg, about0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30 mg/kg, about0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.1 to about 20mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20mg/kg. Values and ranges intermediate to the recited values are alsointended to be part of this invention.

For example, the antisense polynucleotide agent may be administered at adose of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1,9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 2, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50mg/kg. Values and ranges intermediate to the recited values are alsointended to be part of this invention.

In another embodiment, the antisense polynucleotide agent isadministered at a dose of about 0.5 to about 50 mg/kg, about 0.75 toabout 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kgb,about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45mg/kg, about 20 to about 45 mg/kg, about to about 45 mg/kg, about 25 toabout 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg,about 40 to about 45 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 toabout 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb,about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40mg/kg, about 35 to about 40 mg/kg, about 0.5 to about 30 mg/kg, about0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5to about 30 mg/kg, about to about 30 mg/kg, about 15 to about 30 mg/kg,about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 toabout 30 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. In oneembodiment, the antisense polynucleotide agent is administered at a doseof about 10 mg/kg to about 30 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

For example, subjects can be administered, e.g., subcutaneously orintravenously, a single therapeutic amount of antisense polynucleotideagent, such as about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275,0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55,0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825,0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1,9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5,13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5,20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5,27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Valuesand ranges intermediate to the recited values are also intended to bepart of this invention.

In some embodiments, subjects are administered, e.g., subcutaneously orintravenously, multiple doses of a therapeutic amount of antisensepolynucleotide agent, such as a dose about 0.1, 0.125, 0.15, 0.175, 0.2,0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475,0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75,0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25,25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50mg/kg. A multi-dose regimine may include administration of a therapeuticamount of antisense polynucleotide agent daily, such as for two days,three days, four days, five days, six days, seven days, or longer.

In other embodiments, subjects are administered, e.g., subcutaneously orintravenously, a repeat dose of a therapeutic amount of antisensepolynucleotide agent, such as a dose about 0.1, 0.125, 0.15, 0.175, 0.2,0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475,0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75,0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25,25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50mg/kg. A repeat-dose regimine may include administration of atherapeutic amount of antisense polynucleotide agent on a regular basis,such as every other day, every third day, every fourth day, twice aweek, once a week, every other week, or once a month.

The pharmaceutical composition can be administered by intravenousinfusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or about a 25minute period. The administration may be repeated, for example, on aregular basis, such as weekly, biweekly (i.e., every two weeks) for onemonth, two months, three months, four months or longer. After an initialtreatment regimen, the treatments can be administered on a less frequentbasis. For example, after administration weekly or biweekly for threemonths, administration can be repeated once per month, for six months ora year or longer.

The pharmaceutical composition can be administered once daily, or theantisense polynucleotide agent can be administered as two, three, ormore sub-doses at appropriate intervals throughout the day or even usingcontinuous infusion or delivery through a controlled releaseformulation. In that case, the antisense polynucleotide agent containedin each sub-dose must be correspondingly smaller in order to achieve thetotal daily dosage. The dosage unit can also be compounded for deliveryover several days, e.g., using a conventional sustained releaseformulation which provides sustained release of the antisensepolynucleotide agent over a several day period. Sustained releaseformulations are well known in the art and are particularly useful fordelivery of agents at a particular site, such as could be used with theagents of the present invention. In this embodiment, the dosage unitcontains a corresponding multiple of the daily dose.

In other embodiments, a single dose of the pharmaceutical compositionscan be long lasting, such that subsequent doses are administered at notmore than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4week intervals. In some embodiments of the invention, a single dose ofthe pharmaceutical compositions of the invention is administered onceper week. In other embodiments of the invention, a single dose of thepharmaceutical compositions of the invention is administered bi-monthly.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual antisense polynucleotide agentsencompassed by the invention can be made using conventionalmethodologies or on the basis of in vivo testing using an appropriateanimal model, as described elsewhere herein.

The pharmaceutical compositions of the present invention can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration.

The antisense polynucleotide agent can be delivered in a manner totarget a particular tissue, such as the liver (e.g., the hepatocytes ofthe liver).

Pharmaceutical compositions and formulations for topical administrationcan include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like can be necessary or desirable. Coated condoms, gloves and thelike can also be useful. Suitable topical formulations include those inwhich the antisense polynucleotide agents featured in the invention arein admixture with a topical delivery agent such as lipids, liposomes,fatty acids, fatty acid esters, steroids, chelating agents andsurfactants. Suitable lipids and liposomes include neutral (e.g.,dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.,dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.,dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). Antisense polynucleotide agents featured in theinvention can be encapsulated within liposomes or can form complexesthereto, in particular to cationic liposomes. Alternatively, antisensepolynucleotide agents can be complexed to lipids, in particular tocationic lipids. Suitable fatty acids and esters include but are notlimited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid,caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid,linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein,dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, anacylcarnitine, an acylcholine, or a C₁₋₂₀ alkyl ester (e.g.,isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceuticallyacceptable salt thereof). Topical formulations are described in detailin U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

A. Antisense Polynucleotide Agent Formulations Comprising MembranousMolecular Assemblies

An antisense polynucleotide agent for use in the compositions andmethods of the invention can be formulated for delivery in a membranousmolecular assembly, e.g., a liposome or a micelle. As used herein, theterm “liposome” refers to a vesicle composed of amphiphilic lipidsarranged in at least one bilayer, e.g., one bilayer or a plurality ofbilayers. Liposomes include unilamellar and multilamellar vesicles thathave a membrane formed from a lipophilic material and an aqueousinterior. The aqueous portion contains the antisense polynucleotideagent composition. The lipophilic material isolates the aqueous interiorfrom an aqueous exterior, which typically does not include the antisensepolynucleotide agent composition, although in some examples, it may.Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomal bilayer fuses with bilayer of the cellular membranes. Asthe merging of the liposome and cell progresses, the internal aqueouscontents that include the antisense polynucleotide agent are deliveredinto the cell where the antisense polynucleotide agent can specificallybind to a target RNA and can mediate antisense inhibition. In some casesthe liposomes are also specifically targeted, e.g., to direct theantisense polynucleotide agent to particular cell types.

A liposome containing an antisense polynucleotide agent can be preparedby a variety of methods. In one example, the lipid component of aliposome is dissolved in a detergent so that micelles are formed withthe lipid component. For example, the lipid component can be anamphipathic cationic lipid or lipid conjugate. The detergent can have ahigh critical micelle concentration and may be nonionic. Exemplarydetergents include cholate, CHAPS, octylglucoside, deoxycholate, andlauroyl sarcosine. The antisense polynucleotide agent preparation isthen added to the micelles that include the lipid component. Thecationic groups on the lipid interact with the antisense polynucleotideagent and condense around the antisense polynucleotide agent to form aliposome. After condensation, the detergent is removed, e.g., bydialysis, to yield a liposomal preparation of antisense polynucleotideagent.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also be adjusted to favorcondensation.

Methods for producing stable polynucleotide delivery vehicles, whichincorporate a polynucleotide/cationic lipid complex as structuralcomponents of the delivery vehicle, are further described in, e.g., WO96/37194, the entire contents of which are incorporated herein byreference. Liposome formation can also include one or more aspects ofexemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad.Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678;Bangham, et al. M Mol. Biol. 23:238, 1965; Olson, et al. Biochim.Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75:4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, etal. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al.Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be usedwhen consistently small (50 to 200 nm) and relatively uniform aggregatesare desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). Thesemethods are readily adapted to packaging antisense polynucleotide agentpreparations into liposomes.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged nucleicacid molecules to form a stable complex. The positively charged nucleicacid/liposome complex binds to the negatively charged cell surface andis internalized in an endosome. Due to the acidic pH within theendosome, the liposomes are ruptured, releasing their contents into thecell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleicacids rather than complex with it. Since both the nucleic acid and thelipid are similarly charged, repulsion rather than complex formationoccurs. Nevertheless, some nucleic acid is entrapped within the aqueousinterior of these liposomes. pH-sensitive liposomes have been used todeliver nucleic acids encoding the thymidine kinase gene to cellmonolayers in culture. Expression of the exogenous gene was detected inthe target cells (Zhou et al., Journal of Controlled Release, 1992, 19,269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO93/24640; WO 91/16024; Felgner, J Biol. Chem. 269:2550, 1994; Nabel,Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649,1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver antisense polynucleotide agents to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated antisense polynucleotide agents in their internalcompartments from metabolism and degradation (Rosoff, in “PharmaceuticalDosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p.245). Important considerations in the preparation of liposomeformulations are the lipid surface charge, vesicle size and the aqueousvolume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of Antisense polynucleotideagent (see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMAand its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration; liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer an antisense polynucleotide agent into the skin.In some implementations, liposomes are used for delivering antisensepolynucleotide agents to epidermal cells and also to enhance thepenetration of antisense polynucleotide agents into dermal tissues,e.g., into skin. For example, the liposomes can be applied topically.Topical delivery of drugs formulated as liposomes to the skin has beendocumented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992,vol. 2,405-410 and du Plessis et al., Antiviral Research, 18, 1992,259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690,1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth.Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth.Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad.Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith antisense polynucleotide agents are useful for treating adermatological disorder.

Liposomes that include antisense polynucleotide agent can be made highlydeformable. Such deformability can enable the liposomes to penetratethrough pore that are smaller than the average radius of the liposome.For example, transfersomes are a type of deformable liposomes.Transferosomes can be made by adding surface edge activators, usuallysurfactants, to a standard liposomal composition. Transfersomes thatinclude antisense polynucleotide agents can be delivered, for example,subcutaneously by infection in order to deliver antisense polynucleotideagents to keratinocytes in the skin. In order to cross intact mammalianskin, lipid vesicles must pass through a series of fine pores, each witha diameter less than 50 nm, under the influence of a suitabletransdermal gradient. In addition, due to the lipid properties, thesetransferosomes can be self-optimizing (adaptive to the shape of pores,e.g., in the skin), self-repairing, and can frequently reach theirtargets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described inU.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008;61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008;61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCTapplication no PCT/US2007/080331, filed Oct. 3, 2007 also describesformulations that are amenable to the present invention.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes can be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, in“Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y.,1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in “Pharmaceutical Dosage Forms”, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

The antisense polynucleotide agent for use in the compositions andmethods of the invention can also be provided as micellar formulations.“Micelles” are defined herein as a particular type of molecular assemblyin which amphipathic molecules are arranged in a spherical structuresuch that all the hydrophobic portions of the molecules are directedinward, leaving the hydrophilic portions in contact with the surroundingaqueous phase. The converse arrangement exists if the environment ishydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the antisensepolynucleotide agent composition, an alkali metal C₈ to C₂₂ alkylsulphate, and a micelle forming compounds. Exemplary micelle formingcompounds include lecithin, hyaluronic acid, pharmaceutically acceptablesalts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract,cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein,monooleates, monolaurates, borage oil, evening of primrose oil, menthol,trihydroxy oxo cholanyl glycine and pharmaceutically acceptable saltsthereof, glycerin, polyglycerin, lysine, polylysine, triolein,polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethersand analogues thereof, chenodeoxycholate, deoxycholate, and mixturesthereof. The micelle forming compounds may be added at the same time orafter addition of the alkali metal alkyl sulphate. Mixed micelles willform with substantially any kind of mixing of the ingredients butvigorous mixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe antisense polynucleotide agent composition and at least the alkalimetal alkyl sulphate. The first micellar composition is then mixed withat least three micelle forming compounds to form a mixed micellarcomposition. In another method, the micellar composition is prepared bymixing the antisense polynucleotide agent composition, the alkali metalalkyl sulphate and at least one of the micelle forming compounds,followed by addition of the remaining micelle forming compounds, withvigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

B. Lipid Particles

Antisense polynucleotide agents of in the invention may be fullyencapsulated in a lipid formulation, e.g., a LNP, or other nucleicacid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipidparticle comprising a lipid layer encapsulating a pharmaceuticallyactive molecule. LNPs typically contain a cationic lipid, a non-cationiclipid, and a lipid that prevents aggregation of the particle (e.g., aPEG-lipid conjugate). LNPs are extremely useful for systemicapplications, as they exhibit extended circulation lifetimes followingintravenous (i.v.) injection and accumulate at distal sites (e.g., sitesphysically separated from the administration site). LNPs include“pSPLP,” which include an encapsulated condensing agent-nucleic acidcomplex as set forth in PCT Publication No. WO 00/03683. The particlesof the present invention typically have a mean diameter of about 50 nmto about 150 nm, more typically about 60 nm to about 130 nm, moretypically about 70 nm to about 110 nm, most typically about 70 nm toabout 90 nm, and are substantially nontoxic. In addition, the nucleicacids when present in the nucleic acid-lipid particles of the presentinvention are resistant in aqueous solution to degradation with anuclease. Nucleic acid-lipid particles and their method of preparationare disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484;6,586,410; 6,815,432; 6,858,225; 8,158,601; and 8,058,069; U.S.Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to antisense polynucleotide agent ratio) will be in the range offrom about 1:1 to about 50:1, from about 1:1 to about 25:1, from about3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recitedranges are also contemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1), or a mixture thereof. The cationic lipid can comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-santisense polynucleotide agent nanoparticles. Synthesisof 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described inU.S. provisional patent application No. 61/107,998 filed on Oct. 23,2008, which is herein incorporated by reference.

In one embodiment, the lipid-antisense polynucleotide agent particleincludes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10%DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particlesize of 63.0+20 nm and a 0.027 antisense polynucleotide agent/LipidRatio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutrallipid including, but not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid can be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles can be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the lipidoid ND98.4HC1 (MW 1487) (see U.S. patentapplication Ser. No. 12/056,230, filed Mar. 26, 2008, which isincorporated herein by reference), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-antisense polynucleotide agent nanoparticles (i.e., LNPO1particles). Stock solutions of each in ethanol can be prepared asfollows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions canthen be combined in a, e.g., 42:48:10 molar ratio. The combined lipidsolution can be mixed with aqueous antisense polynucleotide agent (e.g.,in sodium acetate pH 5) such that the final ethanol concentration isabout 35-45% and the final sodium acetate concentration is about 100-300mM. Lipid-antisense polynucleotide agent nanoparticles typically formspontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNPO1 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-antisense polynucleotide agent formulationsare described in Table 1.

TABLE 1 cationic lipid/non-cationic lipid/cholesterol/PEG-lipidconjugate Ionizable/Cationic Lipid Lipid:santisense polynucleotide agentratio SNALP-1 1,2-Dilinolenyloxy-N,N-dimethylaminopropaneDLinDMA/DPPC/Cholesterol/PEG-cDMA (DLinDMA) (57.1/7.1/34.4/1.4)lipid:santisense polynucleotide agent ~7:1 2-XTC2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DPPC/Cholesterol/PEG-cDMAdioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:santisense polynucleotide agent~7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5lipid:santisense polynucleotide agent ~6:1 LNP062,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:santisense polynucleotide agent~11:1 LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5,lipid:santisense polynucleotide agent ~6:1 LNP082,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC) 60/7.5/31/1.5, lipid:santisense polynucleotide agent~11:1 LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 50/10/38.5/1.5Lipid:santisense polynucleotide agent 10:1 LNP10(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-ALN100/DSPC/Cholesterol/PEG-DMG octadeca-9,12-dienyl)tetrahydro-3aH-50/10/38.5/1.5 cyclopenta[d][1,3]dioxol-5-amine (ALN100)Lipid:santisense polynucleotide agent 10:1 LNP11(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- MC-3/DSPC/Cholesterol/PEG-DMGtetraen-19-yl 4-(dimethylamino)butanoate 50/10/38.5/1.5 (MC3)Lipid:santisense polynucleotide agent 10:1 LNP121,1′-(2-(4-(2-((2-(bis(2- Tech G1/DSPC/Cholesterol/PEG-DMGhydroxydodecyl)amino)ethyl)(2- 50/10/38.5/1.5hydroxydodecyl)amino)ethyl)piperazin-1- Lipid:santisense polynucleotideagent 10:1 yl)ethylazanediyl)didodecan-2-ol (Tech G1) LNP13 XTCXTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:santisense polynucleotideagent: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:santisensepolynucleotide agent: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:santisensepolynucleotide agent: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG50/10/38.5/1.5 Lipid:santisense polynucleotide agent: 7:1 LNP17 MC3MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:santisense polynucleotideagent: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5Lipid:santisense polynucleotide agent: 12:1 LNP19 MC3MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:santisense polynucleotide agent:8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:santisensepolynucleotide agent: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG50/10/38.5/1.5 Lipid:santisense polynucleotide agent: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:santisense polynucleotideagent: 10:1DSPC: distearoylphosphatidylcholineDPPC: dipalmitoylphosphatidylcholinePEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000)PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg molwt of 2000)PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg molwt of 2000)SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference.

XTC comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No.61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun.10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009;U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, andInternational Application No. PCT/US2010/022614, filed Jan. 29, 2010,which are hereby incorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. Publication No.2010/0324120, filed Jun. 10, 2010, the entire contents of which arehereby incorporated by reference.

ALNY-100 comprising formulations are described, e.g., Internationalpatent application number PCT/US09/63933, filed on Nov. 10, 2009, whichis hereby incorporated by reference.

C12-200 comprising formulations are described in U.S. Provisional Ser.No. 61/175,770, filed May 5, 2009 and International Application No.PCT/US10/33777, filed May 5, 2010, which are hereby incorporated byreference.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders can be desirable. In some embodiments, oralformulations are those in which the antisense polynucleotide agentsfeatured in the invention are administered in conjunction with one ormore penetration enhancer surfactants and chelators. Suitablesurfactants include fatty acids and/or esters or salts thereof, bileacids and/or salts thereof. Suitable bile acids/salts includechenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA),cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid,glycholic acid, glycodeoxycholic acid, taurocholic acid,taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodiumglycodihydrofusidate. Suitable fatty acids include arachidonic acid,undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid,myristic acid, palmitic acid, stearic acid, linoleic acid, linolenicacid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, anacylcholine, or a monoglyceride, a diglyceride or a pharmaceuticallyacceptable salt thereof (e.g., sodium). In some embodiments,combinations of penetration enhancers are used, for example, fattyacids/salts in combination with bile acids/salts. One exemplarycombination is the sodium salt of lauric acid, capric acid and UDCA.Further penetration enhancers include polyoxyethylene-9-lauryl ether,polyoxyethylene-20-cetyl ether. Antisense polynucleotide agents featuredin the invention can be delivered orally, in granular form includingsprayed dried particles, or complexed to form micro or nanoparticles.Antisense polynucleotide agent complexing agents include poly-aminoacids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor antisense polynucleotide agents and their preparation are describedin detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, andU.S. Pat. No. 6,747,014, each of which is incorporated herein byreference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver, e.g., when treatinghepatic disorders, e.g., hepatic carcinoma.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention can also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions can further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and antioxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, LV., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used can be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions ofantisense polynucleotide agents are formulated as microemulsions. Amicroemulsion can be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and DrugDelivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typicallymicroemulsions are systems that are prepared by first dispersing an oilin an aqueous surfactant solution and then adding a sufficient amount ofa fourth component, generally an intermediate chain-length alcohol toform a transparent system. Therefore, microemulsions have also beendescribed as thermodynamically stable, isotropically clear dispersionsof two immiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen,LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions can form spontaneously whentheir components are brought together at ambient temperature. This canbe particularly advantageous when formulating thermolabile drugs,peptides or antisense polynucleotide agents. Microemulsions have alsobeen effective in the transdermal delivery of active components in bothcosmetic and pharmaceutical applications. It is expected that themicroemulsion compositions and formulations of the present inventionwill facilitate the increased systemic absorption of antisensepolynucleotide agents from the gastrointestinal tract, as well asimprove the local cellular uptake of antisense polynucleotide agents andnucleic acids.

Microemulsions of the present invention can also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the antisensepolynucleotide agents of the present invention. Penetration enhancersused in the microemulsions of the present invention can be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

iii. Microparticles

An antisense polynucleotide agent of the invention may be incorporatedinto a particle, e.g., a microparticle. Microparticles can be producedby spray-drying, but may also be produced by other methods includinglyophilization, evaporation, fluid bed drying, vacuum drying, or acombination of these techniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly antisense polynucleotide agents, to the skin of animals.Most drugs are present in solution in both ionized and nonionized forms.However, usually only lipid soluble or lipophilic drugs readily crosscell membranes. It has been discovered that even non-lipophilic drugscan cross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of antisensepolynucleotide agents through the mucosa is enhanced. In addition tobile salts and fatty acids, these penetration enhancers include, forexample, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether andpolyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants andpolymers in drug delivery, Informa Health Care, New York, N.Y., 2002;Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi etal., J. Pharm. Pharmacol., 1988, 40, 252).

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (see e.g., Malmsten,M. Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption ofantisense polynucleotide agents through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents includebut are not limited to disodium ethylenediaminetetraacetate (EDTA),citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylateand homovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. etal., Excipient development for pharmaceutical, biotechnology, and drugdelivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al.,J. Control Rel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of antisense polynucleotide agents through thealimentary mucosa (see e.g., Muranishi, Critical Reviews in TherapeuticDrug Carrier Systems, 1990, 7, 1-33). This class of penetrationenhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J Pharm. Pharmacol., 1987, 39,621-626).

Agents that enhance uptake of antisense polynucleotide agents at thecellular level can also be added to the pharmaceutical and othercompositions of the present invention. For example, cationic lipids,such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationicglycerol derivatives, and polycationic molecules, such as polylysine(Lollo et al., PCT Application WO 97/30731), are also known to enhancethe cellular uptake of antisense polynucleotide agents. Examples ofcommercially available transfection reagents include, for exampleLipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000™(Invitrogen; Carlsbad, Calif.), 293fectin™ (Invitrogen; Carlsbad,Calif.), Cellfectin™ (Invitrogen; Carlsbad, Calif.), DMRIE-C™(Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX (Invitrogen; Carlsbad,Calif.), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, Calif.),Lipofectamine™ (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen;Carlsbad, Calif.), Oligofectamine™ (Invitrogen; Carlsbad, Calif.),Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 TransfectionReagent (Roche; Grenzacherstrasse, Switzerland), DOTAP LiposomalTransfection Reagent (Grenzacherstrasse, Switzerland), DOSPER LiposomalTransfection Reagent (Grenzacherstrasse, Switzerland), or Fugene(Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^(a)D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents can be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

v. Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioated antisense polynucleotide agent in hepatic tissue canbe reduced when it is coadministered with polyinosinic acid, dextransulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense polynucleotide agent Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense polynucleotide agent & Nucl. Acid Drug Dev., 1996, 6,177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions can also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

vii. Other Components

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions can contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or can contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more antisense polynucleotide agents and(b) one or more agents which function by a non-antisense inhibitionmechanism and which are useful in treating a hemolytic disorder.Examples of such agents include, but are not limited to ananti-inflammatory agent, anti-steatosis agent, anti-viral, and/oranti-fibrosis agent. In addition, other substances commonly used toprotect the liver, such as silymarin, can also be used in conjunctionwith the antisense polynucleotide agents described herein. Other agentsuseful for treating liver diseases include telbivudine, entecavir, andprotease inhibitors such as telaprevir and other disclosed, for example,in Tung et al., U.S. Application Publication Nos. 2005/0148548,2004/0167116, and 2003/0144217; and in Hale et al., U.S. ApplicationPublication No. 2004/0127488.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the invention lies generally within arange of circulating concentrations that include the ED₅₀ with little orno toxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC₅₀ (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the antisensepolynucleotide agents featured in the invention can be administered incombination with other known agents effective in treatment ofpathological processes mediated by ALAS1 expression. In any event, theadministering physician can adjust the amount and timing of antisensepolynucleotide agent administration on the basis of results observedusing standard measures of efficacy known in the art or describedherein.

VII. Methods For Inhibiting ALAS1 Expression

The present invention provides methods of inhibiting expression of ALAS1in a cell. The methods include contacting a cell with a polynucleotideagent of the invention, e.g., an antisense polynucleotide agent of theinvention, in an amount effective to inhibit expression of the ALAS1 inthe cell, thereby inhibiting expression of the ALAS1 in the cell.

Contacting of a cell with an antisense polynucleotide agent may be donein vitro or in vivo. Contacting a cell in vivo with the antisensepolynucleotide agent includes contacting a cell or group of cells withina subject, e.g., a human subject, with the antisense polynucleotideagent. Combinations of in vitro and in vivo methods of contacting arealso possible. Contacting may be direct or indirect, as discussed above.Furthermore, contacting a cell may be accomplished via a targetingligand, including any ligand described herein or known in the art. Inpreferred embodiments, the targeting ligand is a carbohydrate moiety,e.g., a GalNAc₃ ligand, or any other ligand that directs the antisensepolynucleotide agent to a site of interest, e.g., the liver of asubject.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating” and other similar terms, andincludes any level of inhibition.

The phrase “inhibiting expression of an ALAS1” is intended to refer toinhibition of expression of any ALAS1 gene (such as, e.g., a mouse ALAS1gene, a rat ALAS1 gene, a monkey ALAS1 gene, or a human ALAS1 gene) aswell as variants or mutants of an ALAS1 gene. Thus, the ALAS1 gene maybe a wild-type ALAS1 gene, a mutant ALAS1 gene, or a transgenic ALAS1gene in the context of a genetically manipulated cell, group of cells,or organism.

“Inhibiting expression of an ALAS1 gene” includes any level ofinhibition of an ALAS1 gene, e.g., at least partial suppression of theexpression of an ALAS1 gene. The expression of the ALAS1 gene may beassessed based on the level, or the change in the level, of any variableassociated with ALAS1 gene expression, e.g., ALAS1 mRNA level or ALAS1protein level. This level may be assessed in an individual cell or in agroup of cells, including, for example, a sample derived from a subject.

Inhibition may be assessed by a decrease in an absolute or relativelevel of one or more variables that are associated with ALAS1 expressioncompared with a control level. The control level may be any type ofcontrol level that is utilized in the art, e.g., a pre-dose baselinelevel, or a level determined from a similar subject, cell, or samplethat is untreated or treated with a control (such as, e.g., buffer onlycontrol or inactive agent control).

In some embodiments of the methods of the invention, expression of anALAS1 gene is inhibited by at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%. at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99%.

Inhibition of the expression of an ALAS1 gene may be manifested by areduction of the amount of mRNA expressed by a first cell or group ofcells (such cells may be present, for example, in a sample derived froma subject) in which an ALAS1 gene is transcribed and which has or havebeen treated (e.g., by contacting the cell or cells with an antisensepolynucleotide agent of the invention, or by administering an antisensepolynucleotide agent of the invention to a subject in which the cellsare or were present) such that the expression of an ALAS1 gene isinhibited, as compared to a second cell or group of cells substantiallyidentical to the first cell or group of cells but which has not or havenot been so treated (control cell(s)). In preferred embodiments, theinhibition is assessed by expressing the level of mRNA in treated cellsas a percentage of the level of mRNA in control cells, using thefollowing formula:

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

Alternatively, inhibition of the expression of an ALAS1 gene may beassessed in terms of a reduction of a parameter that is functionallylinked to ALAS1 gene expression, e.g., levels of porphyrins and/orporphyrin precursors, e.g., ALA and/or PBG. ALAS1 gene silencing may bedetermined in any cell expressing ALAS1, either constitutively or bygenomic engineering, and by any assay known in the art. The liver is themajor site of ALAS1 expression. Other significant sites of expressioninclude the kidneys and the uterus.

Inhibition of the expression of an ALAS1 protein may be manifested by areduction in the level of the ALAS1 protein that is expressed by a cellor group of cells (e.g., the level of protein expressed in a samplederived from a subject). As explained above for the assessment of mRNAsuppression, the inhibition of protein expression levels in a treatedcell or group of cells may similarly be expressed as a percentage of thelevel of protein in a control cell or group of cells.

A control cell or group of cells that may be used to assess theinhibition of the expression of an ALAS1 gene includes a cell or groupof cells that has not yet been contacted with an antisensepolynucleotide agent of the invention. For example, the control cell orgroup of cells may be derived from an individual subject (e.g., a humanor animal subject) prior to treatment of the subject with an antisensepolynucleotide agent.

The level of ALAS1 mRNA that is expressed by a cell or group of cellsmay be determined using any method known in the art for assessing mRNAexpression. In one embodiment, the level of expression of ALAS1 in asample is determined by detecting a transcribed polynucleotide, orportion thereof, e.g., mRNA of the ALAS1 gene. RNA may be extracted fromcells using RNA extraction techniques including, for example, using acidphenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis),RNeasy RNA preparation kits (Qiagen) or PAXgene (PreAnalytix,Switzerland). Typical assay formats utilizing ribonucleic acidhybridization include nuclear run-on assays, RT-PCR, RNase protectionassays (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, insitu hybridization, and microarray analysis.

In one embodiment, the level of expression of ALAS1 is determined usinga nucleic acid probe. The term “probe”, as used herein, refers to anymolecule that is capable of selectively binding to a specific ALAS1.Probes can be synthesized by one of skill in the art, or derived fromappropriate biological preparations. Probes may be specifically designedto be labeled. Examples of molecules that can be utilized as probesinclude, but are not limited to, RNA, DNA, proteins, antibodies, andorganic molecules.

Isolated mRNA can be used in hybridization or amplification assays thatinclude, but are not limited to, Southern or Northern analyses,polymerase chain reaction (PCR) analyses and probe arrays. One methodfor the determination of mRNA levels involves contacting the isolatedmRNA with a nucleic acid molecule (probe) that can hybridize to ALAS1mRNA. In one embodiment, the mRNA is immobilized on a solid surface andcontacted with a probe, for example by running the isolated mRNA on anagarose gel and transferring the mRNA from the gel to a membrane, suchas nitrocellulose. In an alternative embodiment, the probe(s) areimmobilized on a solid surface and the mRNA is contacted with theprobe(s), for example, in an Affymetrix gene chip array. A skilledartisan can readily adapt known mRNA detection methods for use indetermining the level of ALAS1 mRNA.

An alternative method for determining the level of expression of ALAS1in a sample involves the process of nucleic acid amplification and/orreverse transcriptase (to prepare cDNA) of for example mRNA in thesample, e.g., by RT-PCR (the experimental embodiment set forth inMullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany(1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio Technology 6:1197), rolling circle replication (Lizardiet al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. These detectionschemes are especially useful for the detection of nucleic acidmolecules if such molecules are present in very low numbers. Inparticular aspects of the invention, the level of expression of ALAS1 isdetermined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™System).

The expression levels of ALAS1 mRNA may be monitored using a membraneblot (such as used in hybridization analysis such as Northern, Southern,dot, and the like), or microwells, sample tubes, gels, beads or fibers(or any solid support comprising bound nucleic acids). See U.S. Pat.Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which areincorporated herein by reference. The determination of ALAS1 expressionlevel may also comprise using nucleic acid probes in solution.

In preferred embodiments, the level of mRNA expression is assessed usingbranched DNA (bDNA) assays or real time PCR (qPCR). The use of thesemethods is described and exemplified in the Examples presented herein.

The level of ALAS1 protein expression may be determined using any methodknown in the art for the measurement of protein levels. Such methodsinclude, for example, electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions,absorption spectroscopy, a colorimetric assays, spectrophotometricassays, flow cytometry, immunodiffusion (single or double),immunoelectrophoresis, Western blotting, radioimmunoassay (RIA),enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays,electrochemiluminescence assays, and the like.

The term “sample” as used herein refers to a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, lymph, urine,cerebrospinal fluid, saliva, ocular fluids, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes). In preferred embodiments, a “sample derived from asubject” refers to blood or plasma drawn from the subject. In furtherembodiments, a “sample derived from a subject” refers to liver tissuederived from the subject.

In some embodiments of the methods of the invention, the antisensepolynucleotide agent is administered to a subject such that theantisense polynucleotide agent is delivered to a specific site withinthe subject. The inhibition of expression of ALAS1 may be assessed usingmeasurements of the level or change in the level of ALAS1 mRNA or ALAS1protein in a sample derived from fluid or tissue from the specific sitewithin the subject. In preferred embodiments, the site is the liver. Thesite may also be a subsection or subgroup of cells from any one of theaforementioned sites. The site may also include cells that express aparticular type of receptor.

The phrase “contacting a cell with an antisense polynucleotide agent,”as used herein, includes contacting a cell by any possible means.Contacting a cell with an antisense polynucleotide agent includescontacting a cell in vitro with the antisense polynucleotide agent orcontacting a cell in vivo with the antisense polynucleotide agent. Thecontacting may be done directly or indirectly. Thus, for example, theantisense polynucleotide agent may be put into physical contact with thecell by the individual performing the method, or alternatively, theantisense polynucleotide agent may be put into a situation that willpermit or cause it to subsequently come into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the antisense polynucleotide agent. Contacting a cell in vivomay be done, for example, by injecting the antisense polynucleotideagent into or near the tissue where the cell is located, or by injectingthe antisense polynucleotide agent into another area, e.g., thebloodstream or the subcutaneous space, such that the agent willsubsequently reach the tissue where the cell to be contacted is located.For example, the antisense polynucleotide agent may contain and/or becoupled to a ligand, e.g., GalNAc3, that directs the antisensepolynucleotide agent to a site of interest, e.g., the liver.Combinations of in vitro and in vivo methods of contacting are alsopossible. For example, a cell may also be contacted in vitro with anantisense polynucleotide agent and subsequently transplanted into asubject.

In one embodiment, contacting a cell with an antisense polynucleotideagent includes “introducing” or “delivering the antisense polynucleotideagent into the cell” by facilitating or effecting uptake or absorptioninto the cell. Absorption or uptake of an antisense polynucleotide agentcan occur through unaided diffusive or active cellular processes, or byauxiliary agents or devices. Introducing an antisense polynucleotideagent into a cell may be in vitro and/or in vivo. For example, for invivo introduction, antisense polynucleotide agent can be injected into atissue site or administered systemically. In vivo delivery can also bedone by a beta-glucan delivery system, such as those described in U.S.Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No.2005/0281781, the entire contents of which are hereby incorporatedherein by reference. In vitro introduction into a cell includes methodsknown in the art such as electroporation and lipofection. Furtherapproaches are described herein below and/or are known in the art.

VIII. Methods for Treating or Preventing an ALAS1-Associated Disorder

The present invention also provides therapeutic and prophylactic methodswhich include administering to a subject having an ALAS1-associateddisease, e.g., porphyria, an antisense polynucleotide agent orpharmaceutical compositions comprising an antisense polynucleotide agentof the invention. In some aspects of the invention, the methods furtherinclude administering to the subject an additional therapeutic agent,such as glucose and/or a heme product such as hemin.

In one aspect, the present invention provides methods of treating asubject having a disorder that would benefit from reduction in ALAS1expression, e.g., an ALAS1-associated disease, e.g., porphyria. Thetreatment methods (and uses) of the invention include administering tothe subject, e.g., a human, a therapeutically effective amount of anantisense polynucleotide agent targeting an ALAS1 gene or apharmaceutical composition comprising an antisense polynucleotide agenttargeting an ALAS1 gene, thereby treating the subject having a disorderthat would benefit from reduction in ALAS1 expression.

In another aspect, the present invention provides methods of treating asubject having a disorder that would benefit from reduction in an ALAS1expression, e.g., an ALAS1-associated disease, e.g., porphyria, whichinclude administering to the subject, e.g., a human, a therapeuticallyeffective amount of an antisense polynucleotide agent targeting an ALAS1gene or a pharmaceutical composition comprising an antisensepolynucleotide agent targeting an ALAS1 gene, and an additionaltherapeutic agent, such as glucose and/or a heme product such as hemin,thereby treating the subject having a disorder that would benefit fromreduction in ALAS1 expression.

In one aspect, the invention provides methods of preventing at least onesymptom in a subject having a disorder that would benefit from reductionin ALAS1 expression, e.g., an ALAS1-associated disease, e.g., porphyria.The methods include administering to the subject a prophylacticallyeffective amount of an antisense polynucleotide agent targeting an ALAS1gene or a pharmaceutical composition comprising an antisensepolynucleotide agent targeting an ALAS1 gene, thereby preventing atleast one symptom in the subject having a disorder that would benefitfrom reduction in ALAS1 expression.

In another aspect, the invention provides methods of preventing at leastone symptom in a subject having a disorder that would benefit fromreduction in ALAS1 expression, e.g., an ALAS1-associated disease, e.g.,porphyria. The methods include administering to the subject aprophylactically effective amount of an antisense polynucleotide agenttargeting an ALAS1 gene or a pharmaceutical composition comprising anantisense polynucleotide agent targeting an ALAS1 gene, and anadditional therapeutic agent, such as glucose and/or a heme product suchas hemin, thereby preventing at least one symptom in the subject havinga disorder that would benefit from reduction in ALAS1 expression.

As used herein, “an ALAS1 associated disease”, “a disorder related toALAS1 expression,” a “disease related to ALAS1 expression, a“pathological process related to ALAS1 expression,” or the like includesany condition, disorder, or disease in which ALAS1 expression is altered(e.g., elevated), the level of one or more porphyrins is altered (e.g.,elevated), the level or activity of one or more enzymes in the hemebiosynthetic pathway (porphyrin pathway) is altered, or other mechanismsthat lead to pathological changes in the heme biosynthetic pathway. Forexample, an antisense polynucleotide agent targeting an ALAS1 gene, or acombination thereof, may be used for treatment of conditions in whichlevels of a porphyrin or a porphyrin precursor (e.g., ALA or PBG) areelevated (e.g., certain porphyrias), or conditions in which there aredefects in the enzymes of the heme biosynthetic pathway (e.g., certainporphyrias). Disorders related to ALAS1 expression include, for example,X-linked sideroblastic anemia (XLSA), ALA deyhdratase deficiencyporphyria (Doss porphyria), acute intermittent porphyria (AIP),congenital erythropoietic porphyria, prophyria cutanea tarda, hereditarycoproporphyria (coproporphyria), variegate porphyria, erythropoieticprotoporphyria (EPP), and transient erythroporphyria of infancy.

As used herein, a “subject” to be treated according to the methodsdescribed herein, includes a human or non-human animal, e.g., a mammal.The mammal may be, for example, a rodent (e.g., a rat or mouse) or aprimate (e.g., a monkey). In some embodiments, the subject is a human.

In some embodiments, the subject is suffering from a disorder related toALAS1 expression (e.g., has been diagnosed with a porphyria or hassuffered from one or more symptoms of porphyria and is a carrier of amutation associated with porphyria) or is at risk of developing adisorder related to ALAS1 expression (e.g., a subject with a familyhistory of porphyria, or a subject who is a carrier of a geneticmutation associated with porphyria).

Classifications of porphyrias, including acute hepatic porphyrias, aredescribed, e.g., in Balwani, M. & Desnick, R. J., Blood, 120(23),published online as Blood First Edition paper, July 12, 102; DOI10.1182/blood-2012-05-423186. As described in Balwain & Desnick, acuteintermittent porphyria (AIP) hereditary coproporphyria (HCP), variegateporphyria (VP) are autosomal dominant porphyrias and ALA deyhdratasedeficiency porphyria (ADP) is autosomal recessive. In rare cases, AIP,HCP, and VP occur as homozygous dominant forms. In addition, there is arare homozygous recessive form of porphyria cutanea tarda (PCT), whichis the single hepatic cutaneous porphyria, and is also known ashepatoerythropoietic porphyria. The clinical and laboratory features ofthese porphyrias are described in the Table below.

Human hepatic porphyrias: clinical and laboratory features

Enzyme Principal activity, Deficient symptoms, % of Increased porphyrinprecursors and/or porphyrins* Porphyria enzyme Inheritance NV or CPnormal Erythrocytes Urine Stool Acute hepatic porphyrias ADP ALA- AR NV~5 Zn-protoporphyrin ALA, — dehydratase coproporphyrin III AIP HMB- ADNV ~50 — ALA, PBG, — synthase uroporphyrin HCP COPRO- AD NV and CP ~50 —ALA, PBG, coproporphyrin oxidase coproporphyrin III III VP PROTO- AD NVand CP ~50 — ALA, PBG coproporphyrin oxidase coproporphyrin III, IIIprotoporphyrin Hepatic cutaneous porphyrias PCT URO- Sporadic CP <20 —uroporphyrin, uroporphyrin, decarboxylase or AD 7-carboxylate7-carboxylate porphyrin porphyrin AR indicates autosomal recessive; AD,autosomal dominant; NV, neurovisceral; CP, cutaneous photosensitivity;and —, not applicable. *Increases that may be important for diagnosis.

In some embodiments, the subject has or is at risk for developing aporphyria, e.g., a hepatic porphyria, e.g., AIP, HCP, VP, ADP, orhepatoerythropoietic porphyria.

In some embodiments, the porphyria is an acute hepatic porphyria, e.g.,an acute hepatic porphyria is elected from acute intermittent porphyria(ATP), hereditary coproporphyria (HCP), variegate porphyria (VP), andALA deyhdratase deficiency porphyria (ADP).

In some embodiments, the porphyria is a dual porphyria, e.g., at leasttwo porphyrias. In some embodiments, the dual porphyria comprises two ormore porphyrias selected from acute intermittent porphyria (AIP)hereditary coproporphyria (HCP), variegate porphyria (VP), and ALAdeyhdratase deficiency porphyria (ADP).

In some embodiments, the porphyria is a homozygous dominant hepaticporphyria (e.g., homozygous dominant AIP, HCP, or VP) orhepatoerythropoietic porphyria. In some embodiments, the porphyria isAIP, HCP, VP, or hepatoerythropoietic porphyria, or a combinationthereof (e.g., a dual porphyria). In embodiments, the ATP, HCP, or VP iseither heterozygous dominant or homozygous dominant.

In embodiments, the subject has or is at risk for developing aporphyria, e.g., ADP, and shows an elevated level (e.g., an elevatedurine level) of ALA and/or coproporphyrin III. In embodiments, thesubject has or is at risk for developing a porphyria, e.g., ADP, andshows an elevated level of erythrocyte Zn-protoporphyrin.

In embodiments, the subject has or is at risk for developing aporphyria, e.g., AIP, and shows an elevated level (e.g., an elevatedurine level) of ALA, PBG, and/or uroporphyrin.

In embodiments, the subject has or is at risk for developing aporphyria, e.g., HCP, and shows an elevated level (e.g., an elevatedurine level) of ALA, PBG, and/or coproporphyrin III. In embodiments, thesubject has or is at risk for developing a porphyria, e.g., HCP, andshows an elevated level (e.g., an elevated stool level) ofcoproporphyrin III.

In embodiments, the subject has or is at risk for developing aporphyria, e.g., VP, and shows an elevated level (e.g., an elevatedurine level) of ALA, PBG, and/or coproporphyrin III.

In embodiments, the subject has or is at risk for developing aporphyria, e.g., HCP, and shows an elevated level (e.g., an elevatedstool level) of coproporphyrin III and/or protoporphyrin.

In embodiments, the subject has or is at risk for developing aporphyria, e.g., PCT, (e.g., hepatoerythropoietic porphyria) and showsan elevated level (e.g., an elevated urine level) of uroporphyrin and/or7-carboxylate porphyrin. In embodiments, the subject has or is at riskfor developing a porphyria, e.g., PCT, (e.g., hepatoerythropoieticporphyria) and shows an elevated level (e.g., an elevated stool level)of uroporphyrin and/or 7-carboxylate porphyrin.

A mutation associated with porphyria includes any mutation in a geneencoding an enzyme in the heme biosynthetic pathway (porphyrin pathway)or a gene which alters the expression of a gene in the heme biosyntheticpathway. In many embodiments, the subject carries one or more mutationsin an enzyme of the porphyrin pathway (e.g., a mutation in ALAdeydratase or PBG deaminase). In some embodiments, the subject issuffering from an acute porphyria (e.g., AIP, ALA deydratase deficiencyporphyria).

In some cases, patients with an acute hepatic porphyria (e.g., AIP), orpatients who carry mutations associated with an acute hepatic porphyria(e.g., ATP) but who are asymptomatic, have elevated ALA and/or PBGlevels compared with healthy individuals. See, e.g., Floderus, Y. et al,Clinical Chemistry, 52(4): 701-707, 2006; Sardh et al., ClinicalPharmacokinetics, 46(4): 335-349, 2007. In such cases, the level of ALAand/or PBG can be elevated even when the patient is not having, or hasnever had, an attack. In some such cases, the patient is otherwisecompletely asymptomatic. In some such cases, the patient suffers frompain, e.g., neuropathic pain, which can be chronic pain (e.g., chronicneuropathic pain). In some cases, the patient has a neuropathy. In somecases, the patient has a progressive neuropathy.

In some embodiments, the subject to be treated according to the methodsdescribed herein has an elevated level of a porphyrin or a porphyrinprecursor, e.g., ALA and/or PBG. Levels of a porphyrin or a porphyrinprecursor can be assessed using methods known in the art or methodsdescribed herein. For example, methods of assessing urine and plasma ALAand PBG levels, as well as urine and plasma porphyrin levels, aredisclosed in Floderus, Y. et al, Clinical Chemistry, 52(4): 701-707,2006; and Sardh et al., Clinical Pharmacokinetics, 46(4): 335-349, 2007,the entire contents of which are hereby incorporated in their entirety.

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an antisense polynucleotide agent that, whenadministered to a subject having an ALAS1-associated disease, issufficient to effect treatment of the disease (e.g., by diminishing,ameliorating or maintaining the existing disease or one or more symptomsof disease). The “therapeutically effective amount” may vary dependingon the antisense polynucleotide agent, how the agent is administered,the disease and its severity and the history, age, weight, familyhistory, genetic makeup, the types of preceding or concomitanttreatments, if any, and other individual characteristics of the subjectto be treated.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of an antisense polynucleotide agent that, whenadministered to a subject having an ALAS1-associate disease but not yet(or currently) experiencing or displaying symptoms of the disease,and/or a subject at risk of developing an ALAS1-associated disease,e.g., porphyria, is sufficient to prevent or ameliorate the disease orone or more symptoms of the disease. Ameliorating the disease includesslowing the course of the disease or reducing the severity oflater-developing disease. The “prophylactically effective amount” mayvary depending on the antisense polynucleotide agent, how the agent isadministered, the degree of risk of disease, and the history, age,weight, family history, genetic makeup, the types of preceding orconcomitant treatments, if any, and other individual characteristics ofthe patient to be treated.

A “therapeutically effective amount” or “prophylactically effectiveamount” also includes an amount of an antisense polynucleotide agentthat produces some desired local or systemic effect at a reasonablebenefit/risk ratio applicable to any treatment. Antisense polynucleotideagents employed in the methods of the present invention may beadministered in a sufficient amount to produce a reasonable benefit/riskratio applicable to such treatment.

In another aspect, the present invention provides uses of atherapeutically effective amount of an antisense polynucleotide agent ofthe invention for treating a subject, e.g., a subject that would benefitfrom a reduction and/or inhibition of ALAS1 expression.

In another aspect, the present invention provides uses of atherapeutically effective amount of an antisense polynucleotide agent ofthe invention and an additional therapeutic agent, such as glucoseand/or a heme product such as hemin, for treating a subject, e.g., asubject that would benefit from a reduction and/or inhibition of ALAS1expression.

In yet another aspect, the present invention provides use of anantisense polynucleotide agent of the invention targeting an ALAS1 geneor a pharmaceutical composition comprising an antisense polynucleotideagent targeting an ALAS1 gene in the manufacture of a medicament fortreating a subject, e.g., a subject that would benefit from a reductionand/or inhibition of ALAS1 expression, such as a subject having adisorder that would benefit from reduction in ALAS1 expression, e.g.,porphyria.

In another aspect, the present invention provides uses of an antisensepolynucleotide agent of the invention targeting an ALAS1 gene or apharmaceutical composition comprising an antisense polynucleotide agenttargeting an ALAS1 gene in the manufacture of a medicament for use incombination with an additional therapeutic agent, such as glucose and/ora heme product such as hemin, for treating a subject, e.g., a subjectthat would benefit from a reduction and/or inhibition of ALAS1expression, e.g., an ALAS1-associated disease, e.g., porphyria.

In another aspect, the invention provides uses of an antisensepolynucleotide agent of the invention for preventing at least onesymptom in a subject suffering from a disorder that would benefit from areduction and/or inhibition of ALAS1 expression, such as anALAS1-associated disease, e.g., porphyria.

In yet another aspect, the invention provides uses of an antisensepolynucleotide agent of the invention, and an additional therapeuticagent, such as glucose and/or a heme product such as hemin, forpreventing at least one symptom in a subject suffering from a disorderthat would benefit from a reduction and/or inhibition of ALAS1expression, such as an ALAS1-associated disease, e.g., porphyria.

In a further aspect, the present invention provides uses of an antisensepolynucleotide agent of the invention in the manufacture of a medicamentfor preventing at least one symptom in a subject suffering from adisorder that would benefit from a reduction and/or inhibition of ALAS1expression, such as an ALAS1-associated disease, e.g., porphyria.

In a further aspect, the present invention provides uses of an antisensepolynucleotide agent of the invention in the manufacture of a medicamentfor use in combination with an additional therapeutic agent, such asglucose and/or a heme product such as hemin, for preventing at least onesymptom in a subject suffering from a disorder that would benefit from areduction and/or inhibition of ALAS1 expression, such as anALAS1-associated disease, e.g., porphyria.

In one embodiment, an antisense polynucleotide agent targeting ALAS1 isadministered to a subject having an ALAS1-associated disease such thatALAS1 levels, e.g., in a cell, tissue, blood, urine or other tissue orfluid of the subject are reduced by at least about 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or at least about 99% or more and, subsequently, an additionaltherapeutic (as described below) is administered to the subject.

The additional therapeutic may be glucose and/or a heme product such ashemin. The additional therapeutic may be administered to the subject atthe same time as the antisense polynucleotide agent targeting ALAS1 orat a different time.

Moreover, the additional therapeutic may be administered to the subjectin the same formulation as the antisense polynucleotide agent targetingALAS1 or in a different formulation as the antisense polynucleotideagent targeting ALAS1.

The methods and uses of the invention include administering acomposition described herein such that expression of the target ALAS1gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18,24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80hours. In one embodiment, expression of the target ALAS1 gene isdecreased for an extended duration, e.g., at least about two, three,four, five, six, seven days or more, e.g., about one week, two weeks,three weeks, or about four weeks or longer.

Administration of the antisense polynucleotide agent according to themethods and uses of the invention may result in a reduction of theseverity, signs, symptoms, and/or markers of such diseases or disordersin a patient with an ALAS1-associated disease. By “reduction” in thiscontext is meant a statistically significant decrease in such level. Thereduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, orabout 100%.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters, e.g., aplasma or urine level of ALA and/or PBG. Comparisons of the laterreadings with the initial readings provide a physician an indication ofwhether the treatment is effective. It is well within the ability of oneskilled in the art to monitor efficacy of treatment or prevention bymeasuring any one of such parameters, or any combination of parameters.In connection with the administration of an antisense polynucleotideagent targeting ALAS1 or pharmaceutical composition thereof, “effectiveagainst” an ALAS1-associated disease indicates that administration in aclinically appropriate manner results in a beneficial effect for atleast a statistically significant fraction of patients, such asimprovement of symptoms, a cure, a reduction in disease, extension oflife, improvement in quality of life, or other effect generallyrecognized as positive by medical doctors familiar with treating anALAS1-associated disease and the related causes.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given antisense polynucleotide agent drug orformulation of that drug can also be judged using an experimental animalmodel for the given disease as known in the art. When using anexperimental animal model, efficacy of treatment is evidenced when astatistically significant reduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a reduction in theseverity of disease as determined by one skilled in the art of diagnosisbased on a clinically accepted disease severity grading scale. Anypositive change resulting in e.g., lessening of severity of diseasemeasured using the appropriate scale, represents adequate treatmentusing an antisense polynucleotide agent or antisense polynucleotideagent formulation as described herein.

Subjects can be administered a therapeutic amount of antisensepolynucleotide agent, such as about 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg,0.04 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg,0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.45 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.6mg/kg, 0.65 mg/kg, 0.7 mg/kg, 0.75 mg/kg, 0.8 mg/kg, 0.85 mg/kg, 0.9mg/kg, 0.95 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg,2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7mg/kg, 2.8 mg/kg, 2.9 mg/kg, 3.0 mg/kg, 3.1 mg/kg, 3.2 mg/kg, 3.3 mg/kg,3.4 mg/kg, 3.5 mg/kg, 3.6 mg/kg, 3.7 mg/kg, 3.8 mg/kg, 3.9 mg/kg, 4.0mg/kg, 4.1 mg/kg, 4.2 mg/kg, 4.3 mg/kg, 4.4 mg/kg, 4.5 mg/kg, 4.6 mg/kg,4.7 mg/kg, 4.8 mg/kg, 4.9 mg/kg, 5.0 mg/kg, 5.1 mg/kg, 5.2 mg/kg, 5.3mg/kg, 5.4 mg/kg, 5.5 mg/kg, 5.6 mg/kg, 5.7 mg/kg, 5.8 mg/kg, 5.9 mg/kg,6.0 mg/kg, 6.1 mg/kg, 6.2 mg/kg, 6.3 mg/kg, 6.4 mg/kg, 6.5 mg/kg, 6.6mg/kg, 6.7 mg/kg, 6.8 mg/kg, 6.9 mg/kg, 7.0 mg/kg, 7.1 mg/kg, 7.2 mg/kg,7.3 mg/kg, 7.4 mg/kg, 7.5 mg/kg, 7.6 mg/kg, 7.7 mg/kg, 7.8 mg/kg, 7.9mg/kg, 8.0 mg/kg, 8.1 mg/kg, 8.2 mg/kg, 8.3 mg/kg, 8.4 mg/kg, 8.5 mg/kg,8.6 mg/kg, 8.7 mg/kg, 8.8 mg/kg, 8.9 mg/kg, 9.0 mg/kg, 9.1 mg/kg, 9.2mg/kg, 9.3 mg/kg, 9.4 mg/kg, 9.5 mg/kg, 9.6 mg/kg, 9.7 mg/kg, 9.8 mg/kg,9.9 mg/kg, 9.0 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg,35 mg/kg, 40 mg/kg, 45 mg/kg, or about 50 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In certain embodiments, for example, when a composition of the inventioncomprises a antisense polynucleotide agent as described herein and alipid, subjects can be administered a therapeutic amount of antisensepolynucleotide agent, such as about 0.01 mg/kg to about 5 mg/kg, about0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg to about 5 mg/kg, about0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3mg/kg to about 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kgto about 10 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg toabout 10 mg/kg, about 2 mg/kg to about about 2.5 mg/kg, about 2 mg/kg toabout 10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 10mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 10mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6 mg/kg to about 10mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7 mg/kg to about 10mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8 mg/kg to about 10mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to about 10mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

For example, the antisense polynucleotide agent may be administered at adose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10mg/kg. Values and ranges intermediate to the recited values are alsointended to be part of this invention.

In other embodiments, for example, when a composition of the inventioncomprises a antisense polynucleotide agent as described herein and anN-acetylgalactosamine, subjects can be administered a therapeutic amountof antisense polynucleotide agent, such as a dose of about 0.1 to about50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg,about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 toabout 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg,about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50mg/kg, about 35 to about 50 mg/kg, about to about 50 mg/kg, about 45 toabout 50 mg/kg, about 0.1 to about 45 mg/kg, about 0.25 to about 45mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about toabout 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg,about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 toabout 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg,about 40 to about 45 mg/kg, about 0.1 to about 40 mg/kg, about 0.25 toabout 40 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35to about 40 mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.1to about 20 mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about15 to about 20 mg/kg. In one embodiment, when a composition of theinvention comprises a antisense polynucleotide agent as described hereinand an N-acetylgalactosamine, subjects can be administered a therapeuticamount of about 10 to about 30 mg/kg of antisense polynucleotide agent.Values and ranges intermediate to the recited values are also intendedto be part of this invention.

For example, subjects can be administered a therapeutic amount ofantisense polynucleotide agent, such as about 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5,15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5,22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5,29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

The antisense polynucleotide agent can be administered by intravenousinfusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about a 25 minuteperiod. The administration may be repeated, for example, on a regularbasis, such as weekly, biweekly (i.e., every two weeks) for one month,two months, three months, four months or longer. After an initialtreatment regimen, the treatments can be administered on a less frequentbasis. For example, after administration weekly or biweekly for threemonths, administration can be repeated once per month, for six months ora year or longer.

Administration of the antisense polynucleotide agent can reduce ALAS1levels, e.g., in a cell, tissue, blood, urine or other compartment ofthe patient by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%4, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54% 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or at least about 99% or more.

Before administration of a full dose of the antisense polynucleotideagent, patients can be administered a smaller dose, such as a 5%infusion, and monitored for adverse effects, such as an allergicreaction. In another example, the patient can be monitored for unwantedimmunostimulatory effects, such as increased cytokine (e.g., TNF-alphaor INF-alpha) levels.

Owing to the inhibitory effects on ALAS1 expression, a compositionaccording to the invention or a pharmaceutical composition preparedtherefrom can enhance the quality of life.

An antisense polynucleotide agent of the invention may be administeredin “naked” form, or as a “free antisense polynucleotide agent.” A nakedantisense polynucleotide agent is administered in the absence of apharmaceutical composition. The naked antisense polynucleotide agent maybe in a suitable buffer solution. The buffer solution may compriseacetate, citrate, prolamine, carbonate, or phosphate, or any combinationthereof. In one embodiment, the buffer solution is phosphate bufferedsaline (PBS). The pH and osmolarity of the buffer solution containingthe antisense polynucleotide agent can be adjusted such that it issuitable for administering to a subject.

Alternatively, an antisense polynucleotide agent of the invention may beadministered as a pharmaceutical composition, such as an antisensepolynucleotide agent liposomal formulation.

Subjects that would benefit from a reduction and/or inhibition of anALAS1 gene expression are those having an ALAS1-associated disease ordisorder as described herein. In one embodiment, a subject having anALAS1-associated disease has X-linked sideroblastic anemia (XLSA). Inanother embodiment, a subject having an ALAS1-associated disease has ALAdeyhdratase deficiency porphyria (Doss porphyria or ADP). In anotherembodiment, a subject having an ALAS1-associated disease has acuteintermittent porphyria (ATP). In yet another embodiment, a subjecthaving an ALAS1-associated disease has congenital erythropoieticporphyria (CEP). In one embodiment, a subject having an ALAS1-associateddisease has prophyria cutanea tarda (PCT). In another embodiment, asubject having an ALAS1-associated disease has hereditary coproporphyria(coproporphyria, or HCP). In yet another embodiment, a subject having anALAS1-associated disease has variegate porphyria (VP). In oneembodiment, a subject having an ALAS1-associated disease haserythropoietic protoporphyria (EPP). In another embodiment, a subjecthaving an ALAS-associated disease has transient erythroporphyria ofinfancy. In another embodiment, a subject having an ALAS1-associateddisease has hepatic porphyria, e.g., ALA deyhdratase deficiencyporphyria (ADP), AIP, HCP, or VP. In yet another embodiment, a subjecthaving an ALAS1-associated disease has homozygous dominant hepaticporphyria (e.g., homozygous dominant AIP, HCP, or VP. In one embodiment,a subject having an ALAS1-associated disease has hepatoerythropoieticporphyria. In one embodiment, a subject having an ALAS1-associateddisease has dual porphyria.

Treatment of a subject that would benefit from a reduction and/orinhibition of an ALAS1 gene expression includes therapeutic andprophylactic (e.g., the subject is to undergo sensitized (or allogenic)transplant surgery) treatment.

The invention further provides methods and uses of an antisensepolynucleotide agent or a pharmaceutical composition thereof fortreating a subject that would benefit from reduction and/or inhibitionof ALAS1 expression, e.g., a subject having an ALAS1-associated disease,in combination with other pharmaceuticals and/or other therapeuticmethods, e.g., with known pharmaceuticals and/or known therapeuticmethods, such as, for example, those which are currently employed fortreating these disorders. For example, in certain embodiments, anantisense polynucleotide agent targeting ALAS1 is administered incombination with, e.g., an agent useful in treating an ALAS1-associateddisease as described elsewhere herein.

The antisense polynucleotide agent and an additional therapeutic agentand/or treatment may be administered at the same time and/or in the samecombination, e.g., parenterally, or the additional therapeutic agent canbe administered as part of a separate composition or at separate timesand/or by another method known in the art or described herein.

The present invention also provides methods of using an antisensepolynucleotide agent of the invention and/or a composition containing anantisense polynucleotide agent of the invention to reduce and/or inhibitALAS1 expression in a cell. In other aspects, the present inventionprovides an antisense polynucleotide agent of the invention and/or acomposition comprising an antisense polynucleotide agent of theinvention for use in reducing and/or inhibiting ALAS1 expression in acell. In yet other aspects, use of an antisense polynucleotide agent ofthe invention and/or a composition comprising an antisensepolynucleotide agent of the invention for the manufacture of amedicament for reducing and/or inhibiting ALAS1 expression in a cell areprovided.

The methods and uses include contacting the cell with an antisensepolynucleotide agent, e.g., a antisense polynucleotide agent, of theinvention and maintaining the cell for a time sufficient to obtainantisense inhibition of an ALAS1 gene, thereby inhibiting expression ofthe ALAS1 gene in the cell.

Reduction in gene expression can be assessed by any methods known in theart. For example, a reduction in the expression of ALAS1 may bedetermined by determining the mRNA expression level of ALAS1 usingmethods routine to one of ordinary skill in the art, e.g., Northernblotting, qRT-PCR, by determining the protein level of ALAS1 usingmethods routine to one of ordinary skill in the art, such as Westernblotting, immunological techniques, flow cytometry methods, ELISA,and/or by determining a biological activity of ALAS1.

In the methods and uses of the invention the cell may be contacted invitro or in vivo, i.e., the cell may be within a subject. In embodimentsof the invention in which the cell is within a subject, the methods mayinclude further contacting the cell with glucose and/or a heme productsuch as hemin.

A cell suitable for treatment using the methods of the invention may beany cell that expresses an ALAS1 gene. A cell suitable for use in themethods and uses of the invention may be a mammalian cell, e.g., aprimate cell (such as a human cell or a non-human primate cell, e.g., amonkey cell or a chimpanzee cell), a non-primate cell (such as a cowcell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell,a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, adog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bearcell, or a buffalo cell), a bird cell (e.g., a duck cell or a goosecell), or a whale cell. In one embodiment, the cell is a human cell,e.g., a human liver cell.

ALAS1 expression may be inhibited in the cell by at least about 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%.

The in vivo methods and uses of the invention may include administeringto a subject a composition containing an antisense polynucleotide agent,where the antisense polynucleotide agent includes a nucleotide sequencethat is complementary to at least a part of an RNA transcript of theALAS1 gene of the mammal to be treated. When the organism to be treatedis a mammal such as a human, the composition can be administered by anymeans known in the art including, but not limited to subcutaneous,intravenous, oral, intraperitoneal, or parenteral routes, includingintracranial (e.g., intraventricular, intraparenchymal and intrathecal),intramuscular, transdermal, airway (aerosol), nasal, rectal, and topical(including buccal and sublingual) administration. In certainembodiments, the compositions are administered by subcutaneous orintravenous infusion or injection.

In some embodiments, the administration is via a depot injection. Adepot injection may release the antisense polynucleotide agent in aconsistent way over a prolonged time period. Thus, a depot injection mayreduce the frequency of dosing needed to obtain a desired effect, e.g.,a desired inhibition of ALAS1, or a therapeutic or prophylactic effect.A depot injection may also provide more consistent serum concentrations.Depot injections may include subcutaneous injections or intramuscularinjections. In preferred embodiments, the depot injection is asubcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. Inpreferred embodiments, the infusion pump is a subcutaneous infusionpump. In other embodiments, the pump is a surgically implanted pump thatdelivers the antisense polynucleotide agent to the liver.

The mode of administration may be chosen based upon whether local orsystemic treatment is desired and based upon the area to be treated. Theroute and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods forinhibiting the expression of an ALAS1 gene in a mammal, e.g., a human.The present invention also provides a composition comprising anantisense polynucleotide agent that targets an ALAS1 gene in a cell of amammal for use in inhibiting expression of the ALAS1 gene in the mammal.In another aspect, the present invention provides use of an antisensepolynucleotide agent that targets an ALAS1 gene in a cell of a mammal inthe manufacture of a medicament for inhibiting expression of the ALAS1gene in the mammal.

The methods and uses include administering to the mammal, e.g., a human,a composition comprising an antisense polynucleotide agent that targetsan ALAS1 gene in a cell of the mammal and maintaining the mammal for atime sufficient to obtain antisense inhibition of the mRNA transcript ofthe ALAS1 gene, thereby inhibiting expression of the ALAS1 gene in themammal. In some embodiment, the methods further comprise administeringglucose and/or a heme product such as hemin to the subject.

Reduction in gene expression can be assessed by any methods known it theart and by methods, e.g. qRT-PCR, described herein. Reduction in proteinproduction can be assessed by any methods known it the art and bymethods, e.g., ELISA or Western blotting, described herein. In oneembodiment, a puncture liver biopsy sample serves as the tissue materialfor monitoring the reduction in ALAS1 gene and/or protein expression. Inanother embodiment, a blood sample serves as the tissue material formonitoring the reduction in ALAS1 gene and/or protein expression. Inother embodiments, inhibition of the expression of an ALAS1 gene ismonitored indirectly by, for example, determining the expression and/oractivity of a gene in an ALAS1 pathway. Suitable assays are furtherdescribed in the Examples section below.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The entire contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures and the Sequence Listing, arehereby incorporated herein by reference.

EXAMPLES Example 1. Antisense Synthesis

The antisense polynucleotides targeting ALAS1 were synthesized usingstandard synthesis methods well known in the art.

A detailed list of antisense molecules targeting ALAS1 is shown inTables 3 and 4 below.

TABLE 2 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) AAdenosine-3′-phosphate Af 2′-fluoroadenosine-3′-phosphate Afs2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioatea 2′-O-methyladenosine-3′-phosphate as2′-O-methyladenosine-3′-phosphorothioate C cytidine-3′-phosphate dA2′-deoxyadenosine-3′-phosphate dAs 2′-deoxyadenosine-3′-phosphorothioateCf 2′-fluorocytidine-3′-phosphate Cfs2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate c2′-O-methylcytidine-3′-phosphate cs 2′-O-methylcytidine-3′-phosphorothioate dC 2′-deoxycytidine-3′-phosphate dCs2′-deoxycytidine-3′-phosphorothioate G guanosine-3′-phosphate Gf2′-fluoroguanosine-3′-phosphate Gfs2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioateg 2′-O-methylguanosine-3′-phosphate gs2′-O-methylguanosine-3′-phosphorothioate dG2′-deoxyguanosine-3′-phosphate dGs 2′-deoxyguanosine-3′-phosphorothioateT 5′-methyluridine-3′-phosphate Tf2′-fluoro-5-methyluridine-3′-phosphate Tfs2′-fluoro-5-methyluridine-3′-phosphorothioate Ts5-methyluridine-3′-phosphorothioate t2′-O-methyl-5-methyluridine-3′-phosphate ts2′-O-methyl-5-methyluridine-3′-phosphorothioate dT2′-deoxythymidine-3′-phosphate dTs 2′-deoxythymidine-3′-phosphorothioateU Uridine-3′-phosphate Uf 2′-fluorouridine-3′-phosphate Ufs2′-fluorouridine-3′-phosphorothioate Us uridine-3′-phosphorothioate u2′-O-methyluridine-3′-phosphate us2′-O-methyluridine-3′-phosphorothioate dU 2′-deoxyuridine-3′-phosphatedUs 2′-deoxyuridine-3′-phosphorothioate s phosphorothioate linkage N anynucleotide (G, A, C, T or U) L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol Hyp-(GalNAc-alkyl)3 (dt) deoxy-thymine (5MdC) or (m5dC)5′-methyl-deoxycytidine-3′-phosphate (5MdC)s or5′-methyl-deoxycytidine-3′-phosphorothioate (m5dCs)

TABLE 3Antisense polynucleotides targeting aminolevulinic acid synthase-1 (ALAS1)SEQ Alternative ID Sequence ID Sequence ID Modified Sequence (5′-3′) NO:A-130452.1 X10361gsusgsascs(5MdC)sdGs(5MdC)sdTsdGs(5MdC)sdGs(5MdC)sdAsdTsdGsgscsgscsc 7A-130453.1 X10362usascsasgs(5MdC)sdGsdGsdGsdAsdGsdTsdGsdAs(5MdC)s(5MdC)scsgscsusg 8A-130454.1 X10363csgscscsusdTsdAsdAsdTsdAsdTsdAs(5MdC)sdAsdGs(5MdC)scsgsgsgsa 9A-130455.1 X10364csgsasuscsdGs(5MdC)s(5MdC)sdGsdGs(5MdC)sdGs(5MdC)s(5MdC)sdTsdTsusasasusa10 A-130456.1 X10365cscsuscsasdGsdGs(5MdC)s(5MdC)sdGs(5MdC)sdGsdAsdTs(5MdC)sdGsgscscsgsg 11A-130457.1 X10366cscsgsgsgsdAsdGs(5MdC)sdAsdGs(5MdC)s(5MdC)sdTs(5MdC)sdAsdGsgsgscscsg 12A-130458.1 X10367ususgscscs(5MdC)sdTsdTsdGsdTs(5MdC)s(5MdC)sdGsdGsdGsdAsasgscsasg 13A-130459.1 X10368gsasasascsdGs(5MdC)sdTs(5MdC)sdGsdTsdTsdGs(5MdC)s(5MdC)s(5MdC)scsususgsu14 A-130460.1 X10369asasgsuscs(5MdC)sdAsdAsdAs(5MdC)sdGsdAsdAsdAs(5MdC)sdGsgscsuscsg 15A-130461.1 X10370uscsasasgsdTs(5MdC)sdGsdAsdGsdAsdAsdGsdTs(5MdC)s(5MdC)scsasasasc 16A-130462.1 X10371asgsgscsgsdGsdGs(5MdC)sdAs(5MdC)sdTs(5MdC)sdAsdAsdGsdTsuscsgsasg 17A-130463.1 X10372gscsgsgscsdGsdAsdAsdGsdGsdAsdGsdGs(5MdC)sdGsdGsgsgscsasc 18 A-130464.1X10373 usgscsasgsdAsdGsdGs(5MdC)sdGsdGs(5MdC)sdGsdGs(5MdC)sdGsgsasasgsg19 A-130465.1 X10374csgscsusgsdAsdGsdGsdAs(5MdC)sdTsdGs(5MdC)sdAsdGsdAsasgsgscsg 20A-130466.1 X10375gsgscsasusdAsdAs(5MdC)sdTsdGs(5MdC)sdGs(5MdC)sdTsdGsdAsasgsgsasc 21A-130467.1 X10376gsgsasasgsdAsdAs(5MdC)sdTsdGsdGsdGs(5MdC)sdAsdTsdAsasascsusg 22A-130468.1 X10377cscscscsas(5MdC)sdAsdGs(5MdC)sdGsdGsdGsdAsdAsdGsdAsasascsusg 23A-130469.1 X10378gsusgsgsus(5MdC)sdGsdTsdGsdTs(5MdC)s(5MdC)s(5MdC)s(5MdC)sdAs(5MdC)scsasgscsg24 A-130470.1 X10379gsgsasusus(5MdC)s(5MdC)sdTs(5MdC)s(5MdC)sdGsdTsdGsdGsdTs(5MdC)scsgsusgsu25 A-130471.1 X10380cscsusgsasdAsdGs(5MdC)sdAsdAsdGsdGsdAsdTsdTs(5MdC)scscsuscsc 26A-130472.1 X10381gsuscscscsdGsdAsdGsdTs(5MdC)s(5MdC)s(5MdC)sdTsdGsdAsdAsasgscsasa 27A-130473.1 X10382gsuscscsasdGs(5MdC)sdAsdGsdGsdGsdTs(5MdC)s(5MdC)s(5MdC)sdGsgsasgsusc 28A-130474.1 X10383csgsasgsgsdAsdAsdGsdGsdGsdGsdTs(5MdC)s(5MdC)sdAsdGsgscsasgsg 29A-130475.1 X10384cscscscsusdAsdAsdAs(5MdC)s(5MdC)s(5MdC)sdGsdAsdGsdGsdAsasasgsgsg 30A-130476.1 X10385gsuscscscs(5MdC)sdAs(5MdC)sdAsdTs(5MdC)s(5MdC)s(5MdC)s(5MdC)sdTsdAsasasascsc31 A-130477.1 X10386csusususcsdTs(5MdC)s(5MdC)sdTsdGsdGsdTs(5MdC)s(5MdC)s(5MdC)s(5MdC)scsascsasu32 A-130478.1 X10387gsgsgsasus(5MdC)s(5MdC)sdTsdGsdAs(5MdC)sdTsdTsdTs(5MdC)sdTsuscscsusg 33A-130479.1 X10388asasgsascsdTs(5MdC)sdTsdTsdAsdGsdGsdGsdAsdTs(5MdC)scscsusgsa 34A-130480.1 X10389cscsasgsgs(5MdC)sdAsdGsdGsdGsdAsdAsdGsdAs(5MdC)sdTsuscsususa 35A-130481.1 X10390ascsuscsasdTs(5MdC)s(5MdC)sdAsdTs(5MdC)s(5MdC)sdAsdGsdGs(5MdC)scsasgsgsg36 A-130482.1 X10391asgsasasgsdAsdAsdGs(5MdC)s(5MdC)sdAs(5MdC)sdTs(5MdC)sdAsdTsuscscsasu 37A-130483.1 X10392 asuscsusasdGsdGsdTsdGsdGsdAsdGsdAsdAsdGsdAsasasgscsc38 A-130484.1 X10393usgsusgsgsdAsdAsdAsdGsdAsdAsdTs(5MdC)sdTsdAsdGsgsgsusgsg 39 A-130485.1X10394 usgscsusgsdGs(5MdC)sdTs(5MdC)s(5MdC)sdTsdGsdTsdGsdGsdAsasasasgsa40 A-130486.1 X10395uscsasgsgsdAsdAsdGsdTsdAsdTsdGs(5MdC)sdTsdGsdGsgscsuscsc 41 A-130487.1X10396 csuscsuscs(5MdC)sdAsdTsdGsdTsdTs(5MdC)sdAsdGsdGsdAsasasgsusa 42A-130488.1 X10397gscsgsasas(5MdC)sdAsdAs(5MdC)sdAs(5MdC)sdTs(5MdC)sdTs(5MdC)s(5MdC)scsasusgsu43 A-130489.1 X10398asusgsgsgs(5MdC)sdAsdGs(5MdC)sdGsdGs(5MdC)sdGsdAsdAs(5MdC)scsasascsa 44A-130490.1 X10399csgsgsgsasdTsdAsdAsdGsdAsdAsdTsdGsdGsdGs(5MdC)scsasgscsg 45 A-130491.1X10400 csusgsgsgsdGsdGsdAs(5MdC)sdTs(5MdC)sdGsdGsdGsdAsdTsusasasgsa 46A-130492.1 X10401gscsasgsasdAsdAsdGsdGs(5MdC)s(5MdC)sdTsdGsdGsdGsdGsgsgsascsu 47A-130493.1 X10402cscsusgscsdTsdTsdTs(5MdC)sdTsdGs(5MdC)sdAsdGsdAsdAsasasgsgsc 48A-130494.1 X10403csasgsasgsdAsdTsdTsdTsdGs(5MdC)s(5MdC)sdTsdGs(5MdC)sdTsusususcsu 49A-130495.1 X10404csasusasgsdAsdAs(5MdC)sdAsdAs(5MdC)sdAsdGsdAsdGsdAsasusususg 50A-130496.1 X10405csasgsususdTsdTsdGsdGsdGs(5MdC)sdAsdTsdAsdGsdAsasascsasa 51 A-130497.1X10406 csasuscsusdTsdGsdGsdGsdGs(5MdC)sdAsdGsdTsdTsdTsususgsgsg 52A-130498.1 X10407csasascsusdTs(5MdC)s(5MdC)sdAsdTs(5MdC)sdAsdTs(5MdC)sdTsdTsusgsgsgsg 53A-130499.1 X10408gsgscsususdGsdGs(5MdC)s(5MdC)s(5MdC)s(5MdC)sdAsdAs(5MdC)sdTsdTsuscscsasu54 A-130500.1 X10409cscsgsasgsdGsdGsdGs(5MdC)sdTsdGsdGs(5MdC)sdTsdTsdGsgsgscscsc 55A-130501.1 X10410usgsgsascsdAsdAsdTsdGs(5MdC)s(5MdC)s(5MdC)sdGsdAsdGsdGsgsgsgscsu 56A-130502.1 X10411ascsusgscsdTsdGs(5MdC)sdAsdGsdTsdGsdGsdAs(5MdC)sdAsasasusgsc 57A-130503.1 X10412ususgsgsusdAsdGsdTsdGsdTsdAs(5MdC)sdTsdGs(5MdC)sdTsusgscsasg 58A-130504.1 X10413csusususgsdAsdTs(5MdC)sdTsdGsdTsdTsdGsdGsdTsdAsasgsusgsu 59 A-130505.1X10414 gsgsasgsgsdGsdGsdTsdTsdTs(5MdC)sdTsdTsdTsdGsdAsasuscsusg 60A-130506.1 X10415csuscsascsdTsdGsdGs(5MdC)s(5MdC)sdGsdGsdAsdGsdGsdGsgsgsususu 61A-130507.1 X10416ususususgsdTs(5MdC)sdTsdTsdTs(5MdC)sdTs(5MdC)sdAs(5MdC)sdTsusgsgscsc 62A-130508.1 X10417gscscsususdAsdGs(5MdC)sdAsdGsdTsdTsdTsdTsdGsdTsuscsususu 63 A-130509.1X10418ususgsgsas(5MdC)s(5MdC)sdTsdTsdGsdGs(5MdC)s(5MdC)sdTsdTsdAsasgscsasg 64A-130510.1 X10419csasgsgsasdGsdTs(5MdC)sdTsdGsdTsdTsdGsdGsdAs(5MdC)scscsususg 65A-130511.1 X10420usgsgsgsasdTs(5MdC)s(5MdC)sdAsdTs(5MdC)sdAsdGsdGsdAsdGsgsuscsusg 66A-130512.1 X10421usgsgsascsdTs(5MdC)sdTsdGs(5MdC)sdTsdGsdGsdGsdAsdTsuscscsasu 67A-130513.1 X10422gsusgsusgs(5MdC)s(5MdC)sdAsdTs(5MdC)sdTsdGsdGsdAs(5MdC)sdTsuscsusgsc 68A-130514.1 X10423gsascsgsgsdAsdAsdGs(5MdC)sdTsdGsdTsdGsdTsdGs(5MdC)scscsasusc 69A-130515.1 X10424gsgsgsgsusdGsdTs(5MdC)s(5MdC)sdAsdGsdAs(5MdC)sdGsdGsdAsasasgscsu 70A-130516.1 X10425usgsgscsasdGsdGs(5MdC)sdAsdAsdGsdGsdGsdGsdTsdGsgsuscscsa 71 A-130517.1X10426 cscscsusgsdGs(5MdC)sdTsdTsdGsdTsdGsdGs(5MdC)sdAsdGsgsgscsasa 72A-130518.1 X10427gscsususgs(5MdC)sdAsdGsdTsdGs(5MdC)s(5MdC)s(5MdC)sdTsdGsdGsgscsususg 73A-130519.1 X10428asasgsgsgs(5MdC)sdAsdTsdTsdTsdGs(5MdC)sdTsdTsdGs(5MdC)scsasgsusg 74A-130520.1 X10429gscsusgscs(5MdC)sdAsdGsdGsdAsdAsdAsdGsdGsdGs(5MdC)scsasususu 75A-130521.1 X10430asususcsasdTs(5MdC)sdTsdGsdTsdGs(5MdC)sdTsdGs(5MdC)s(5MdC)scsasgsgsa 76A-130522.1 X10431usgscscsus(5MdC)sdTs(5MdC)sdTsdGsdAsdTsdTs(5MdC)sdAsdTsuscsusgsu 77A-130523.1 X10432asasgsascsdAs(5MdC)sdTsdGs(5MdC)sdTsdGs(5MdC)s(5MdC)sdTs(5MdC)scsuscsusg78 A-130524.1 X10433gsgscsususdTsdGs(5MdC)sdAsdGsdAsdAsdGsdAs(5MdC)sdAsascsusgsc 79A-130525.1 X10434gscsuscsasdAsdGsdAs(5MdC)sdTsdGsdGs(5MdC)sdTsdTsdTsusgscsasg 80A-130526.1 X10435uscscsuscs(5MdC)sdTsdGsdAsdAsdGs(5MdC)sdTs(5MdC)sdAsdAsasgsascsu 81A-130527.1 X10436ususcscsusdGs(5MdC)sdAs(5MdC)sdAsdTs(5MdC)s(5MdC)sdTs(5MdC)s(5MdC)scsusgsasa82 A-130528.1 X10437csgsgscsasdTsdTs(5MdC)sdAsdTsdTsdTs(5MdC)s(5MdC)sdTsdGsgscsascsa 83A-130529.1 X10438uscsususus(5MdC)s(5MdC)sdTs(5MdC)sdAs(5MdC)sdGsdGs(5MdC)sdAsdTsususcsasu84 A-130530.1 X10439ususcsasgs(5MdC)sdAsdAs(5MdC)s(5MdC)sdTs(5MdC)sdTsdTsdTs(5MdC)scscsuscsa85 A-130531.1 X10440csusgscsusdGsdAsdGsdGsdTsdTsdTs(5MdC)sdAsdGs(5MdC)scsasascsc 86A-130532.1 X10441ascsascsusdGsdGsdGsdGs(5MdC)s(5MdC)sdTsdGs(5MdC)sdTsdGsgsasgsgsu 87A-130533.1 X10442csascsascsdTsdAsdAs(5MdC)s(5MdC)sdAs(5MdC)sdAs(5MdC)sdTsdGsgsgsgsgsc 88A-130534.1 X10443csasuscsgsdGsdTsdTsdTsdTs(5MdC)sdAs(5MdC)sdAs(5MdC)sdTsusasascsc 89A-130535.1 X10444gsgsasuscs(5MdC)s(5MdC)s(5MdC)sdTs(5MdC)s(5MdC)sdAsdTs(5MdC)sdGsdGsgsusususu90 A-130536.1 X10445csasgsuscs(5MdC)sdAs(5MdC)sdTsdGsdGsdGsdAsdTs(5MdC)s(5MdC)scscscsusc 91A-130537.1 X10446asgsususcsdTsdTs(5MdC)sdAsdGs(5MdC)sdAsdGsdTs(5MdC)s(5MdC)scsascsusg 92A-130538.1 X10447asusgsuscs(5MdC)sdTsdGsdGsdAsdAsdGsdTsdTs(5MdC)sdTsususcsasg 93A-130539.1 X10448csususususdGs(5MdC)sdAsdTsdGsdAsdTsdGsdTs(5MdC)s(5MdC)scsusgsgsa 94A-130540.1 X10449csusgsgsus(5MdC)sdTsdTsdTsdGs(5MdC)sdTsdTsdTsdTsdGsgscsasusg 95A-130541.1 X10450uscsusgsgsdTs(5MdC)sdTsdTsdTsdGs(5MdC)sdTsdTsdTsdTsusgscsasu 96A-130542.1 X10451ususcsusgsdGsdTs(5MdC)sdTsdTsdTsdGs(5MdC)sdTsdTsdTsususgscsa 97A-130543.1 X10452usususcsusdGsdGsdTs(5MdC)sdTsdTsdTsdGs(5MdC)sdTsdTsusususgsc 98A-130544.1 X10453csusususcsdTsdGsdGsdTs(5MdC)sdTsdTsdTsdGs(5MdC)sdTsususususg 99A-130545.1 X10454uscsususus(5MdC)sdTsdGsdGsdTs(5MdC)sdTsdTsdTsdGs(5MdC)scsusususu 100A-130546.1 X10455csuscsususdTs(5MdC)sdTsdGsdGsdTs(5MdC)sdTsdTsdTsdGsgscsususu 101A-130547.1 X10456ascsuscsusdTsdTs(5MdC)sdTsdGsdGsdTs(5MdC)sdTsdTsdTsusgscsusu 102A-130548.1 X10457csascsuscsdTsdTsdTs(5MdC)sdTsdGsdGsdTs(5MdC)sdTsdTsususgscsu 103A-130549.1 X10458ascsascsus(5MdC)sdTsdTsdTs(5MdC)sdTsdGsdGsdTs(5MdC)sdTsusususgsc 104A-130550.1 X10459gsascsascsdTs(5MdC)sdTsdTsdTs(5MdC)sdTsdGsdGsdTs(5MdC)scsusususg 105A-130551.1 X10460asgsascsas(5MdC)sdTs(5MdC)sdTsdTsdTs(5MdC)sdTsdGsdGsdTsuscsususu 106A-130552.1 X10461gsasgsascsdAs(5MdC)sdTs(5MdC)sdTsdTsdTs(5MdC)sdTsdGsdGsgsuscsusu 107A-130553.1 X10462usgsasgsas(5MdC)sdAs(5MdC)sdTs(5MdC)sdTsdTsdTs(5MdC)sdTsdGsgsgsuscsu 108A-130554.1 X10463asusgsasgsdAs(5MdC)sdAs(5MdC)sdTs(5MdC)sdTsdTsdTs(5MdC)sdTsusgsgsusc 109A-130555.1 X10464gsasusgsasdGsdAs(5MdC)sdAs(5MdC)sdTs(5MdC)sdTsdTsdTs(5MdC)scsusgsgsu 110A-130556.1 X10465asgsasusgsdAsdGsdAs(5MdC)sdAs(5MdC)sdTs(5MdC)sdTsdTsdTsuscsusgsg 111A-130557.1 X10466asasgsasusdGsdAsdGsdAs(5MdC)sdAs(5MdC)sdTs(5MdC)sdTsdTsususcsusg 112A-130558.1 X10467gsasasgsasdTsdGsdAsdGsdAs(5MdC)sdAs(5MdC)sdTs(5MdC)sdTsusususcsu 113A-130559.1 X10468asgsasasgsdAsdTsdGsdAsdGsdAs(5MdC)sdAs(5MdC)sdTs(5MdC)scsusususc 114A-130560.1 X10469asasgsasasdGsdAsdTsdGsdAsdGsdAs(5MdC)sdAs(5MdC)sdTsuscsususu 115A-130561.1 X10470gsasasgsasdAsdGsdAsdTsdGsdAsdGsdAs(5MdC)sdAs(5MdC)scsuscsusu 116A-130562.1 X10471usgsasasgsdAsdAsdGsdAsdTsdGsdAsdGsdAs(5MdC)sdAsascsuscsu 117 A-130563.1X10472 ususgsasasdGsdAsdAsdGsdAsdTsdGsdAsdGsdAs(5MdC)scsascsusc 118A-130564.1 X10473 csususgsasdAsdGsdAsdAsdGsdAsdTsdGsdAsdGsdAsascsascsu119 A-130565.1 X10474uscsususgsdAsdAsdGsdAsdAsdGsdAsdTsdGsdAsdGsgsascsasc 120 A-130566.1X10475 asuscsususdGsdAsdAsdGsdAsdAsdGsdAsdTsdGsdAsasgsascsa 121A-130567.1 X10476 usasuscsusdTsdGsdAsdAsdGsdAsdAsdGsdAsdTsdGsgsasgsasc122 A-130568.1 X10477ususasuscsdTsdTsdGsdAsdAsdGsdAsdAsdGsdAsdTsusgsasgsa 123 A-130569.1X10478 gsususasus(5MdC)sdTsdTsdGsdAsdAsdGsdAsdAsdGsdAsasusgsasg 124A-130570.1 X10479asgsususasdTs(5MdC)sdTsdTsdGsdAsdAsdGsdAsdAsdGsgsasusgsa 125 A-130571.1X10480 asasgsususdAsdTs(5MdC)sdTsdTsdGsdAsdAsdGsdAsdAsasgsasusg 126A-130572.1 X10481gsasusususdTsdGsdGs(5MdC)sdAsdAsdGsdTsdTsdAsdTsuscsususg 127 A-130573.1X10482 asgsusgsgsdAsdAsdAs(5MdC)sdAsdGsdAsdTsdTsdTsdTsusgsgscsa 128A-130574.1 X10483 csasusascsdTsdGsdAsdAsdAsdAsdGsdTsdGsdGsdAsasasascsa129 A-130575.1 X10484asasgsasasdAs(5MdC)sdGsdAsdTs(5MdC)sdAsdTsdAs(5MdC)sdTsusgsasasa 130A-130576.1 X10485usususususdTs(5MdC)sdTs(5MdC)sdAsdAsdAsdGsdAsdAsdAsascsgsasu 131A-130577.1 X10486uscsuscsasdTs(5MdC)sdAsdAsdTsdTsdTsdTsdTsdTsdTsuscsuscsa 132 A-130578.1X10487 uscsasusus(5MdC)sdTsdTsdTsdTsdTs(5MdC)sdTs(5MdC)sdAsdTsuscsasasu133 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asasususgsdAsdTsdTsdGs(5MdC)sdTsdTsdGs(5MdC)sdAs(5MdC)scsgsusasg237 A-130683.1 X10592ascscsgsusdAsdGsdGsdGsdTsdAsdAsdTsdTsdGsdAsasususgsc 238 A-130684.1X10593 uscscscscsdGsdGsdGsdGs(5MdC)sdAs(5MdC)s(5MdC)sdGsdTsdAsasgsgsgsu239 A-130685.1 X10594gsgsasgscsdTs(5MdC)sdTsdTs(5MdC)sdTs(5MdC)s(5MdC)s(5MdC)s(5MdC)sdGsgsgsgsgsc240 A-130686.1 X10595gscsasasus(5MdC)s(5MdC)sdGsdTsdAsdGsdGsdAsdGs(5MdC)sdTsuscsususc 241A-130687.1 X10596asgsgsgsgsdTsdGsdGsdGsdGsdGs(5MdC)sdAsdAsdTs(5MdC)scscsgsusa 242A-130688.1 X10597 gsusgsusgsdTsdGsdGsdTsdGsdAsdGsdGsdGsdGsdTsusgsgsgsg243 A-130689.1 X10598asuscsasus(5MdC)sdTsdGsdGsdGsdGsdTsdGsdTsdGsdTsusgsgsusg 244 A-130690.1X10599 gsasasgsusdAsdGsdTsdTs(5MdC)sdAsdTs(5MdC)sdAsdTs(5MdC)scsusgsgsg245 A-130691.1 X10600gsasususcsdTs(5MdC)sdAsdAsdGsdGsdAsdAsdGsdTsdAsasgsususc 246 A-130692.1X10601 gsusgsascsdTsdAsdGs(5MdC)sdAsdGsdAsdTsdTs(5MdC)sdTsuscsasasg 247A-130693.1 X10602ususgscsusdTs(5MdC)s(5MdC)sdAsdTsdGsdTsdGsdAs(5MdC)sdTsusasgscsa 248A-130694.1 X10603cscsasgscs(5MdC)s(5MdC)s(5MdC)sdAs(5MdC)sdTsdTsdGs(5MdC)sdTsdTsuscscsasu249 A-130695.1 X10604gsgscsusus(5MdC)sdAsdGsdTsdTs(5MdC)s(5MdC)sdAsdGs(5MdC)s(5MdC)scscscsasc250 A-130696.1 X10605usgsasgsgsdAsdAsdTsdGsdAsdGsdGs(5MdC)sdTsdTs(5MdC)scsasgsusu 251A-130697.1 X10606usgscsascsdTs(5MdC)sdAsdGs(5MdC)sdTsdGsdAsdGsdGsdAsasasusgsa 252A-130698.1 X10607csusgscsasdGsdAsdAsdGsdTsdTsdGs(5MdC)sdAs(5MdC)sdTsuscsasgsc 253A-130699.1 X10608csasgsusgsdGs(5MdC)s(5MdC)sdTs(5MdC)s(5MdC)sdTsdGs(5MdC)sdAsdGsgsasasgsu254 A-130700.1 X10609csususcsasdAsdAsdAsdTsdGs(5MdC)sdAsdGsdTsdGsdGsgscscsusc 255 A-130701.1X10610uscsascsus(5MdC)sdAsdTs(5MdC)sdAs(5MdC)sdTsdTs(5MdC)sdAsdAsasasasusg 256A-130702.1 X10611csususcsus(5MdC)sdTs(5MdC)sdTsdTsdTs(5MdC)sdAs(5MdC)sdTs(5MdC)scsasuscsa257 A-130703.1 X10612asgsasasasdTsdAsdGsdGsdAs(5MdC)sdTsdTs(5MdC)sdTs(5MdC)scsuscsusu 258A-130704.1 X10613csuscsasasdGs(5MdC)s(5MdC)sdTsdGsdAsdGsdAsdAsdAsdTsusasgsgsa 259A-130705.1 X10614usascscsasdAs(5MdC)sdTsdTsdGs(5MdC)sdTs(5MdC)sdAsdAsdGsgscscsusg 260A-130706.1 X10615cscsusgsasdGs(5MdC)sdAsdGsdAsdTsdAs(5MdC)s(5MdC)sdAsdAsascsususg 261A-130707.1 X10616csasusgscsdTs(5MdC)sdAsdGsdGs(5MdC)s(5MdC)sdTsdGsdAsdGsgscsasgsa 262A-130708.1 X10617usasasususdGsdAsdGsdGsdTs(5MdC)sdAsdTsdGs(5MdC)sdTsuscsasgsg 263A-130709.1 X10618 ususasasgsdTsdGsdAsdAsdAsdTsdAsdAsdTsdTsdGsgsasgsgsu264 A-130710.1 X10619usgsgscscsdTsdGsdGsdGsdGsdTsdTsdAsdAsdGsdTsusgsasasa 265 A-130711.1X10620 gsasusasusdGsdAsdTsdAsdAsdTsdGsdGs(5MdC)s(5MdC)sdTsusgsgsgsg 266A-130712.1 X10621asgsascscsdAsdTs(5MdC)sdTsdGsdGsdAsdTsdAsdTsdGsgsasusasa 267 A-130713.1X10622 ascsasascsdTs(5MdC)sdTsdGsdAsdAsdGsdAs(5MdC)s(5MdC)sdAsasuscsusg268 A-130714.1 X10623ascsasusasdTsdAsdAsdAsdGsdAs(5MdC)sdAsdAs(5MdC)sdTsuscsusgsa 269A-130715.1 X10624asascsususdAsdAsdTsdTs(5MdC)sdAs(5MdC)sdAsdTsdAsdTsusasasasg 270A-130716.1 X10625asasusususdAsdAsdTsdAsdTsdAsdAs(5MdC)sdTsdTsdAsasasususc 271 A-130717.1X10626 usasusasgsdAsdTsdTsdAsdAsdAsdAsdTsdTsdTsdAsasasusasu 272A-130718.1 X10627asusgsususdTsdTsdTsdAs(5MdC)sdTsdAsdTsdAsdGsdAsasususasa 273 A-130719.1X10628 ususcscsasdGsdGsdAs(5MdC)sdTsdAsdTsdGsdTsdTsdTsusususasc 274A-130720.1 X10629asasgsasasdTsdTsdTsdAsdTsdTsdTs(5MdC)s(5MdC)sdAsdGsgsgsascsu 275A-130721.1 X10630cscsasususdTsdAsdAsdGs(5MdC)sdAsdAsdGsdAsdAsdTsusususasu 276

TABLE 4Antisense polynucleotides targeting aminolevulinic acid synthase-1 (ALAS1)Start position relative to Reverse Complement of Sequence NM_000688.5Unmodified Sequence Unmodified Sequence ID (SEQ ID NO: 2)Modified Sequence (5′-3′) SEQ ID NO: (5′-3′) SEQ ID NO (5′-3′) SEQ ID NONM_000688.5_20- 20 gsusgsascs(m5dCs)dGs(m5dCs)dTsdG 277 GUGACCGCUGCGCAUG547 GGCGCAUGCGCAGCGG 817 39_aso s(m5dCs)dGs(m5dCs)dAsdTsgscsgscs CGCCUCAC c NM_000688.5_30- 30 usascsasgs(m5dCs)dGsdGsdGsdAsdG 278UACAGCGGGAGUGAC 548 CAGCGGUCACUCCCGC 818 49_asosdTsdGsdAs(m5dCs)csgscsusg CGCUG UGUA NM_000688.5_40- 40csgscscsusdTsdAsdAsd 279 CGCCUUAAUAUACAGC 549 UCCCGCUGUAUAUUAA 81959_aso TsdAsdTsdAs(m5dCs)d GGGA GGCG AsdGscsgsgsgsa NM_000688.5_50- 50csgsasuscsdGs(m5dCs)(m5dCs)dGsdG 280 CGAUCGCCGGCGCCUU 550UAUUAAGGCGCCGGCG 820 69_aso s(m5dCs)dGs(m5dCs)(m5dCs)dTsusas AAUA AUCGasusa NM_000688.5_60- 60 cscsuscsasdGsdGs(m5dCs)(m5dCs)dG 281CCUCAGGCCGCGAUCG 551 CCGGCGAUCGCGGCCU 821 79_asos(m5dCs)dGsdAsdTs(m5dCs)gscscsgsg CCGG GAGG NM_000688.5_70- 70cscsgsgsgsdAsdGs(m5dCs)dAsdGs(m 282 CCGGGAGCAGCCUCAG 552CGGCCUGAGGCUGCUC 822 89_aso 5dCs)(m5dCs)dTs(m5dCs)dAsgsgscsc GCCG CCGGsg NM_000688.5_80- 80 ususgscscs(m5dCs)dTsdTsdGsdTs(m5dCs) 283UUGCCCUUGUCCGGGA 553 CUGCUCCCGGACAAGG 823 99_aso(m5dCs)dGsdGsdGsasgscsasg GCAG GCAA NM_000688.5_90- 90gsasasascsdGs(m5dCs)dTs(m5dCs)dG 284 GAAACGCUCGUUGCCC 554ACAAGGGCAACGAGCG 824 109_aso sdTsdTsdGs(m5dCs)(m5dCs)csususgs UUGU UUUCu NM_000688.5_100- 100 asasgsuscs(m5dCs)dAsdAsdAs(m5dCs) 285AAGUCCAAACGAAAC 555 CGAGCGUUUCGUUUGG 825 119_asodGsdAsdAsdAs(m5dCs)gscsuscsg GCUCG ACUU NM_000688.5_110- 110uscsasasgsdTs(m5dCs)dGsdAsdGsdAs 286 UCAAGUCGAGAAGUC 556GUUUGGACUUCUCGAC 826 129_aso dAsdGsdTs(m5dCs)csasasasc CAAAC UUGANM_000688.5_120- 120 asgsgscsgsdGsdGs(m5dCs)dAs(m5dCs) 287AGGCGGGCACUCAAG 557 CUCGACUUGAGUGCCC 827 139_asodTs(m5dCs)dAsdAsdGsuscsgsasg UCGAG GCCU NM_000688.5_130- 130gscsgsgscsdGsdAsdAsdGsdGsdAsdGs 288 GCGGCGAAGGAGGCG 558 GUGCCCGCCUCCUUCG828 149_aso dGs(m5dCs)dGsgsgscsasc GGCAC CCGC NM_000688.5_140- 140usgscsasgsdAsdGsdGs(m5dCs)dGsdG 289 UGCAGAGGCGGCGGC 559 CCUUCGCCGCCGCCUC829 159_aso s(m5dCs)dGsdGs(m5dCs)gsasasgsg GAAGG UGCA NM_000688.5_150-150 csgscsusgsdAsdGsdGsdAs(m5dCs)dTs 290 CGCUGAGGACUGCAG 560CGCCUCUGCAGUCCUC 830 169_aso dGs(m5dCs)dAsdGsasgsgscsg AGGCG AGCGNM_000688.5_160- 160 gsgscsasusdAsdAs(m5dCs)dTsdGs 291 GGCAUAACUGCGCUG561 GUCCUCAGCGCAGUUA 831 179_aso (m5dCs)dGs(m5dCs)dTsdGsasgsgsasc AGGACUGCC NM_000688.5_170- 170 gsgsasasgsdAsdAs(m5dCs)dTsdGsdGs 292GGAAGAACUGGGCAU 562 CAGUUAUGCCCAGUUC 832 189_asodGs(m5dCs)dAsdTsasascsusg AACUG UUCC NM_000688.5_180- 180cscscscsas(m5dCs)dAsdGs(m5dCs)dG 293 CCCCACAGCGGGAAGA 563CAGUUCUUCCCGCUGU 833 199_aso sdGsdGsdAsdAsdGsasascsusg ACUG GGGGNM_000688.5_190- 190 gsusgsgsus(m5dCs)dGsdTsdGsdTs(m5dCs) 294GUGGUCGUGUCCCCAC 564 CGCUGUGGGGACACGA 834 209_aso(m5dCs)(m5dCs)(m5dCs)dAscsas AGCG CCAC gscsg NM_000688.5_200- 200gsgsasusus(m5dCs)(m5dCs)dTs(m5dCs) 295 GGAUUCCUCCGUGGUC 565ACACGACCACGGAGGA 835 219_aso (m5dCs)dGsdTsdGsdGsdTscsgsusgs GUGU AUCC uNM_000688.5_210- 210 cscsusgsasdAsdGs(m5dCs)dAsdAsdG 296 CCUGAAGCAAGGAUU566 GGAGGAAUCCUUGCUU 836 229_aso sdGsdAsdTsdTscscsuscsc CCUCC CAGGNM_000688.5_220- 220 gsuscscscsdGsdAsdGsdTs(m5dCs)(m5dCs) 297GUCCCGAGUCCCUGAA 567 UUGCUUCAGGGACUCG 837 239_aso(m5dCs)dTsdGsdAsasgscsasa GCAA GGAC NM_000688.5_230- 230gsuscscsasdGs(m5dCs)dAsdGsdGsdG 298 GUCCAGCAGGGUCCCG 568GACUCGGGACCCUGCU 838 249_aso sdTs(m5dCs)(m5dCs)(m5dCs)gsasgsu AGUC GGACsc NM_000688.5_240- 240 csgsasgsgsdAsdAsdGsdGsdGsdGsdTs 299CGAGGAAGGGGUCCA 569 CCUGCUGGACCCCUUC 839 259_aso(m5dCs)(m5dCs)dAsgscsasgsg GCAGG CUCG NM_000688.5_250- 250cscscscsusdAsdAsdAs(m5dCs)(m5dCs) 300 CCCCUAAACCCGAGGA 570CCCUUCCUCGGGUUUA 840 269_aso (m5dCs)dGsdAsdGsdGsasasgsgsg AGGG GGGGNM_000688.5_260- 260 gsuscscscs(m5dCs)dAs(m5dCs)dAsdT 301GUCCCCACAUCCCCUA 571 GGUUUAGGGGAUGUGG 841 279_asos(m5dCs)(m5dCs)(m5dCs)(m5dCs)dT AACC GGAC sasasascsc NM_000688.5_270-270 csusususcsdTs(m5dCs)(m5dCs)dTsdG 302 CUUUCUCCUGGUCCCC 572AUGUGGGGACCAGGAG 842 289_aso sdGsdTs(m5dCs)(m5dCs)(m5dCs)csas ACAU AAAGcsasu NM_000688.5_280- 280 gsgsgsasus(m5dCs)(m5dCs)dTsdGsdA 303GGGAUCCUGACUUUC 573 CAGGAGAAAGUCAGGA 843 299_asos(m5dCs)dTsdTsdTs(m5dCs)uscscsus UCCUG UCCC g NM_000688.5_290- 290asasgsascsdTs(m5dCs)dTsdTsdAsdGs 304 AAGACUCUUAGGGAU 574UCAGGAUCCCUAAGAG 844 309_aso dGsdGsdAsdTscscsusgsa CCUGA UCUUNM_000688.5_300- 300 cscsasgsgs(m5dCs)dAsdGsdGsdGsdA 305 CCAGGCAGGGAAGAC575 UAAGAGUCUUCCCUGC 845 319_aso sdAsdGsdAs(m5dCs)uscsususa UCUUA CUGGNM_000688.5_310- 310 ascsuscsasdTs(m5dCs)(m5dCs)dAsdT 306ACUCAUCCAUCCAGGC 576 CCCUGCCUGGAUGGAU 846 329_asos(m5dCs)(m5dCs)dAsdGsdGscsasgsgs AGGG GAGU g NM_000688.5_320- 320asgsasasgsdAsdAsdGs(m5dCs)(m5dCs) 307 AGAAGAAGCCACUCA 577AUGGAUGAGUGGCUUC 847 339_aso dAs(m5dCs)dTs(m5dCs)dAsuscscsas UCCAU UUCUu NM_000688.5_330- 330 asuscsusasdGsdGsdTsdGsdGsdAsdGs 308AUCUAGGUGGAGAAG 578 GGCUUCUUCUCCACCU 848 349_aso dAsdAsdGsasasgscscAAGCC AGAU NM_000688.5_340- 340 usgsusgsgsdAsdAsdAsdGsdAsdAsdTs 309UGUGGAAAGAAUCUA 579 CCACCUAGAUUCUUUC 849 359_aso (m5dCs)dTsdAsgsgsusgsgGGUGG CACA NM_000688.5_350- 350 usgscsusgsdGs(m5dCs)dTs(m5dCs) 310UGCUGGCUCCUGUGG 580 UCUUUCCACAGGAGCC 850 369_aso(m5dCs)dTsdGsdTsdGsdGsasasasgsa AAAGA AGCA NM_000688.5_360- 360uscsasgsgsdAsdAsdGsdTsdAsdTsdGs 311 UCAGGAAGUAUGCUG 581 GGAGCCAGCAUACUUC851 379_aso (m5dCs)dTsdGsgscsuscsc GCUCC CUGA NM_000688.5_370- 370csuscsuscs(m5dCs)dAsdTsdGsdTsdTs 312 CUCUCCAUGUUCAGGA 582UACUUCCUGAACAUGG 852 389_aso (m5dCs)dAsdGsdGsasasgsusa AGUA AGAGNM_000688.5_380- 380 gscsgsasas(m5dCs)dAsdAs(m5dCs)dA 313GCGAACAACACUCUCC 583 ACAUGGAGAGUGUUGU 853 399_asos(m5dCs)dTs(m5dCs)dTs(m5dCs)csas AUGU UCGC usgsu NM_000688.5_390- 390asusgsgsgs(m5dCs)dAsdGs(m5dCs)d 314 AUGGGCAGCGGCGAA 584 UGUUGUUCGCCGCUGC854 409_aso GsdGs(m5dCs)dGsdAsdAscsasascsa CAACA CCAU NM_000688.5_400-400 csgsgsgsasdTsdAsdAsdGsdAsdAsdTs 315 CGGGAUAAGAAUGGG 585CGCUGCCCAUUCUUAU 855 419_aso dGsdGsdGscsasgscsg CAGCG CCCGNM_000688.5_410- 410 csusgsgsgsdGsdGsdAs(m5dCs)dTs 316 CUGGGGGACUCGGGA586 UCUUAUCCCGAGUCCC 856 429_aso (m5dCs)dGsdGsdGsdAsusasasgsa UAAGA CCAGNM_000688.5_420- 420 gscsasgsasdAsdAsdGsdGs(m5dCs) 317 GCAGAAAGGCCUGGG587 AGUCCCCCAGGCCUUU 857 439_aso (m5dCs)dTsdGsdGsdGsgsgsascsu GGACU CUGCNM_000688.5_430- 430 cscsusgscsdTsdTsdTs(m5dCs)dTsdGs 318CCUGCUUUCUGCAGAA 588 GCCUUUCUGCAGAAAG 858 449_aso(m5dCs)dAsdGsdAsasasgsgsc AGGC CAGG NM_000688.5_440- 440csasgsasgsdAsdTsdTsdTsdGs(m5dCs) 319 CAGAGAUUUGCCUGC 589AGAAAGCAGGCAAAUC 859 459_aso (m5dCs)dTsdGs(m5dCs)usususcsu UUUCU UCUGNM_000688.5_450- 450 csasusasgsdAsdAs(m5dCs)dAsdAs 320 CAUAGAACAACAGAG590 CAAAUCUCUGUUGUUC 860 469_aso (m5dCs)dAsdGsdAsdGsasusususg AUUUG UAUGNM_000688.5_460- 460 csasgsususdTsdTsdGsdGsdGs(m5dCs) 321CAGUUUUGGGCAUAG 591 UUGUUCUAUGCCCAAA 861 479_aso dAsdTsdAsdGsasascsasaAACAA ACUG NM_000688.5_470- 470 csasuscsusdTsdGsdGsdGsdGs(m5dCs) 322CAUCUUGGGGCAGUU 592 CCCAAAACUGCCCCAA 862 489_aso dAsdGsdTsdTsususgsgsgUUGGG GAUG NM_000688.5_480- 480 csasascsusdTs(m5dCs)(m5dCs)dAsdT 323CAACUUCCAUCAUCUU 593 CCCCAAGAUGAUGGAA 863 499_asos(m5dCs)dAsdTs(m5dCs)dTsusgsgsgs GGGG GUUG g NM_000688.5_490- 490gsgscsususdGsdGs(m5dCs)(m5dCs) 324 GGCUUGGCCCCAACUU 594 AUGGAAGUUGGGGCCA864 509_aso (m5dCs)(m5dCs)dAsdAs(m5dCs)dTsusc CCAU AGCC scsasuNM_000688.5_500- 500 cscsgsasgsdGsdGsdGs(m5dCs)dTsdGs 325CCGAGGGGCUGGCUU 595 GGGCCAAGCCAGCCCC 865 519_asodGs(m5dCs)dTsdTsgsgscscsc GGCCC UCGG NM_000688.5_510- 510usgsgsascsdAsdAsdTsdGs(m5dCs) 326 UGGACAAUGCCCGAG 596 AGCCCCUCGGGCAUUG866 529_aso (m5dCs)(m5dCs)dGsdAsdGsgsgsgscsu GGGCU UCCA NM_000688.5_520-520 ascsusgscsdTsdGs(m5dCs)dAsdGsdTs 327 ACUGCUGCAGUGGAC 597GCAUUGUCCACUGCAG 867 539_aso dGsdGsdAs(m5dCs)asasusgsc AAUGC CAGUNM_000688.5_530- 530 ususgsgsusdAsdGsdTsdGsdTsdAs 328 UUGGUAGUGUACUGC598 CUGCAGCAGUACACUA 868 549_aso (m5dCs)dTsdGs(m5dCs)usgscsasg UGCAGCCAA NM_000688.5_540- 540 csusususgsdAsdTs(m5dCs)dTsdGsdTs 329CUUUGAUCUGUUGGU 599 ACACUACCAACAGAUC 869 559_aso dTsdGsdGsdTsasgsusgsuAGUGU AAAG NM_000688.5_550- 550 gsgsasgsgsdGsdGsdTsdTsdTs(m5dCs) 330GGAGGGGUUUCUUUG 600 CAGAUCAAAGAAACCC 870 569_aso dTsdTsdTsdGsasuscsusgAUCUG CUCC NM_000688.5_560- 560 csuscsascsdTsdGsdGs(m5dCs) 331CUCACUGGCCGGAGGG 601 AAACCCCUCCGGCCAG 871 579_aso(m5dCs)dGsdGsdAsdGsdGsgsgsususu GUUU UGAG NM_000688.5_570- 570ususususgsdTs(m5dCs)dTsdTsdTs(m5 332 UUUUGUCUUUCUCAC 602GGCCAGUGAGAAAGAC 872 589_aso dCs)dTs(m5dCs)dAs(m5dCs)usgsgscs UGGCC AAAAc NM_000688.5_580- 580 gscscsususdAsdGs(m5dCs)dAsdGsdTs 333GCCUUAGCAGUUUUG 603 AAAGACAAAACUGCUA 873 599_aso dTsdTsdTsdGsuscsususuUCUUU AGGC NM_000688.5_590- 590 ususgsgsas(m5dCs)(m5dCs)dTsdTsdG 334UUGGACCUUGGCCUU 604 CUGCUAAGGCCAAGGU 874 609_asosdGs(m5dCs)(m5dCs)dTsdTsasgscsas AGCAG CCAA g NM_000688.5_600- 600csasgsgsasdGsdTs(m5dCs)dTsdGsdTs 335 CAGGAGUCUGUUGGA 605CAAGGUCCAACAGACU 875 619_aso dTsdGsdGsdAscscsususg CCUUG CCUGNM_000688.5_610- 610 usgsgsgsasdTs(m5dCs)(m5dCs)dAsdT 336UGGGAUCCAUCAGGA 606 CAGACUCCUGAUGGAU 876 629_asos(m5dCs)dAsdGsdGsdAsgsuscsusg GUCUG CCCA NM_000688.5_620- 620usgsgsascsdTs(m5dCs)dTsdGs(m5dCs) 337 UGGACUCUGCUGGGA 607AUGGAUCCCAGCAGAG 877 639_aso dTsdGsdGsdGsdAsuscscsasu UCCAU UCCANM_000688.5_630- 630 gsusgsusgs(m5dCs)(m5dCs)dAsdTs 338 GUGUGCCAUCUGGAC608 GCAGAGUCCAGAUGGC 878 649_aso (m5dCs)dTsdGsdGsdAs(m5dCs)uscsusgsUCUGC ACAC c NM_000688.5_640- 640 gsascsgsgsdAsdAsdGs(m5dCs)dTsdGs 339GACGGAAGCUGUGUG 609 GAUGGCACACAGCUUC 879 659_aso dTsdGsdTsdGscscsasuscCCAUC CGUC NM_000688.5_650- 650 gsgsgsgsusdGsdTs(m5dCs)(m5dCs)dA 340GGGGUGUCCAGACGG 610 AGCUUCCGUCUGGACA 880 669_asosdGsdAs(m5dCs)dGsdGsasasgscsu AAGCU CCCC NM_000688.5_660- 660usgsgscsasdGsdGs(m5dCs)dAsdAsdG 341 UGGCAGGCAAGGGGU 611 UGGACACCCCUUGCCU881 679_aso sdGsdGsdGsdTsgsuscscsa GUCCA GCCA NM_000688.5_670- 670cscscsusgsdGs(m5dCs)dTsdTsdGsdTs 342 CCCUGGCUUGUGGCAG 612UUGCCUGCCACAAGCC 882 689_aso dGsdGs(m5dCs)dAsgsgscsasa GCAA AGGGNM_000688.5_680- 680 gscsususgs(m5dCs)dAsdGsdTsdGs 343 GCUUGCAGUGCCCUGG613 CAAGCCAGGGCACUGC 883 699_aso (m5dCs)(m5dCs)(m5dCs)dTsdGsgscsusu CUUGAAGC sg NM_000688.5_690- 690 asasgsgsgs(m5dCs)dAsdTsdTsdTsdGs 344AAGGGCAUUUGCUUG 614 CACUGCAAGCAAAUGC 884 709_aso(m5dCs)dTsdTsdGscsasgsusg CAGUG CCUU NM_000688.5_700- 700gscsusgscs(m5dCs)dAsdGsdGsdAsdA 345 GCUGCCAGGAAAGGG 615 AAAUGCCCUUUCCUGG885 719_aso sdAsdGsdGsdGscsasususu CAUUU CAGC NM_000688.5_710- 710asususcsasdTs(m5dCs)dTsdGsdTsdGs 346 AUUCAUCUGUGCUGCC 616UCCUGGCAGCACAGAU 886 729_aso (m5dCs)dTsdGs(m5dCs)csasgsgsa AGGA GAAUNM_000688.5_720- 720 usgscscsus(m5dCs)dTs(m5dCs)dTsdG 347UGCCUCUCUGAUUCAU 617 ACAGAUGAAUCAGAGA 887 739_asosdAsdTsdTs(m5dCs)dAsuscsusgsu CUGU GGCA NM_000688.5_730- 730asasgsascsdAs(m5dCs)dTsdGs(m5dCs) 348 AAGACACUGCUGCCUC 618CAGAGAGGCAGCAGUG 888 749_aso dTsdGs(m5dCs)(m5dCs)dTscsuscsus UCUG UCUU gNM_000688.5_740- 740 gsgscsususdTsdGs(m5dCs)dAsdGsdA 349 GGCUUUGCAGAAGAC619 GCAGUGUCUUCUGCAA 889 759_aso sdAsdGsdAs(m5dCs)ascsusgsc ACUGC AGCCNM_000688.5_750- 750 gscsuscsasdAsdGsdAs(m5dCs)dTsdGs 350GCUCAAGACUGGCUU 620 CUGCAAAGCCAGUCUU 890 769_asodGs(m5dCs)dTsdTsusgscsasg UGCAG GAGC NM_000688.5_760- 760uscscsuscs(m5dCs)dTsdGsdAsdAsdGs 351 UCCUCCUGAAGCUCAA 621AGUCUUGAGCUUCAGG 891 779_aso (m5dCs)dTs(m5dCs)dAsasgsascsu GACU AGGANM_000688.5_770- 770 ususcscsusdGs(m5dCs)dAs(m5dCs)dA 352UUCCUGCACAUCCUCC 622 UUCAGGAGGAUGUGCA 892 789_asosdTs(m5dCs)(m5dCs)dTs(m5dCs)csus UGAA GGAA gsasa NM_000688.5_780- 780csgsgscsasdTsdTs(m5dCs)dAsdTsdTs 353 CGGCAUUCAUUUCCUG 623UGUGCAGGAAAUGAAU 893 799_aso dTs(m5dCs)(m5dCs)dTsgscsascsa CACA GCCGNM_000688.5_790- 790 uscsususus(m5dCs)(m5dCs)dTs(m5dCs) 354UCUUUCCUCACGGCAU 624 AUGAAUGCCGUGAGGA 894 809_asodAs(m5dCs)dGsdGs(m5dCs)dAsusu UCAU AAGA scsasu NM_000688.5_800- 800ususcsasgs(m5dCs)dAsdAs(m5dCs) 355 UUCAGCAACCUCUUUC 625 UGAGGAAAGAGGUUGC895 819_aso (m5dCs)dTs(m5dCs)dTsdTsdTscscsuscsa CUCA UGAANM_000688.5_810- 810 csusgscsusdGsdAsdGsdGsdTsdTsdTs 356 CUGCUGAGGUUUCAG626 GGUUGCUGAAACCUCA 896 829_aso (m5dCs)dAsdGscsasascsc CAACC GCAGNM_000688.5_820- 820 ascsascsusdGsdGsdGsdGs(m5dCs) 357 ACACUGGGGCCUGCUG627 ACCUCAGCAGGCCCCA 897 839_aso (m5dCs)dTsdGs(m5dCs)dTsgsasgsgsu AGGUGUGU NM_000688.5_830- 830 csascsascsdTsdAsdAs(m5dCs)(m5dCs) 358CACACUAACCACACUG 628 GCCCCAGUGUGGUUAG 898 849_asodAs(m5dCs)dAs(m5dCs)dTsgsgsgsgs GGGC UGUG c NM_000688.5_840- 840csasuscsgsdGsdTsdTsdTsdTs(m5dCs) 359 CAUCGGUUUUCACACU 629GGUUAGUGUGAAAACC 899 859_aso dAs(m5dCs)dAs(m5dCs)usasascsc AACC GAUGNM_000688.5_850- 850 gsgsasuscs(m5dCs)(m5dCs)(m5dCs)d 360GGAUCCCCUCCAUCGG 630 AAAACCGAUGGAGGGG 900 869_asoTs(m5dCs)(m5dCs)dAsdTs(m5dCs)d UUUU AUCC Gsgsusususu NM_000688.5_860-860 csasgsuscs(m5dCs)dAs(m5dCs)dTsdG 361 CAGUCCACUGGGAUCC 631GAGGGGAUCCCAGUGG 901 879_aso sdGsdGsdAsdTs(m5dCs)cscscsusc CCUC ACUGNM_000688.5_870- 870 asgsususcsdTsdTs(m5dCs)dAsdGs 362 AGUUCUUCAGCAGUCC632 CAGUGGACUGCUGAAG 902 889_aso (m5dCs)dAsdGsdTs(m5dCs)csascsusg ACUGAACU NM_000688.5_880- 880 asusgsuscs(m5dCs)dTsdGsdGsdAsdAs 363AUGUCCUGGAAGUUC 633 CUGAAGAACUUCCAGG 903 899_asodGsdTsdTs(m5dCs)ususcsasg UUCAG ACAU NM_000688.5_890- 890csususususdGs(m5dCs)dAsdTsdGsdA 364 CUUUUGCAUGAUGUC 634 UCCAGGACAUCAUGCA904 909_aso sdTsdGsdTs(m5dCs)csusgsgsa CUGGA AAAG NM_000688.5_900- 900csusgsgsus(m5dCs)dTsdTsdTsdGs(m5 365 CUGGUCUUUGCUUUU 635CAUGCAAAAGCAAAGA 905 919_aso dCs)dTsdTsdTsdTsgscsasusg GCAUG CCAGNM_000688.5_901- 901 uscsusgsgsdTs(m5dCs)dTsdTsdTsdGs 366UCUGGUCUUUGCUUU 636 AUGCAAAAGCAAAGAC 906 920_aso(m5dCs)dTsdTsdTsusgscsasu UGCAU CAGA NM_000688.5_902- 902ususcsusgsdGsdTs(m5dCs)dTsdTsdTs 367 UUCUGGUCUUUGCUU 637UGCAAAAGCAAAGACC 907 921_aso dGs(m5dCs)dTsdTsususgscsa UUGCA AGAANM_000688.5_903- 903 usususcsusdGsdGsdTs(m5dCs)dTsdTs 368UUUCUGGUCUUUGCU 638 GCAAAAGCAAAGACCA 922_aso dTsdGs(m5dCs)dTsusususgscUUUGC GAAA 908 NM_000688.5_904- 904 csusususcsdTsdGsdGsdTs(m5dCs)dTs 369CUUUCUGGUCUUUGC 639 CAAAAGCAAAGACCAG 909 923_asodTsdTsdGs(m5dCs)ususususg UUUUG AAAG NM_000688.5_905- 905uscsususus(m5dCs)dTsdGsdGsdTs 370 UCUUUCUGGUCUUUG 640 AAAAGCAAAGACCAGA910 924_aso (m5dCs)dTsdTsdTsdGscsusususu CUUUU AAGA NM_000688.5_906- 906csuscsususdTs(m5dCs)dTsdGsdGsdTs 371 CUCUUUCUGGUCUUU 641AAAGCAAAGACCAGAA 911 925_aso (m5dCs)dTsdTsdTsgscsususu GCUUU AGAGNM_000688.5_907- 907 ascsuscsusdTsdTs(m5dCs)dTsdGsdGs 372ACUCUUUCUGGUCUU 642 AAGCAAAGACCAGAAA 912 926_asodTs(m5dCs)dTsdTsusgscsusu UGCUU GAGU NM_000688.5_908- 908csascsuscsdTsdTsdTs(m5dCs)dTsdGs 373 CACUCUUUCUGGUCUU 643AGCAAAGACCAGAAAG 913 927_aso dGsdTs(m5dCs)dTsususgscsu UGCU AGUGNM_000688.5_909- 909 ascsascsus(m5dCs)dTsdTsdTs(m5dCs) 374ACACUCUUUCUGGUCU 644 GCAAAGACCAGAAAGA 914 928_asodTsdGsdGsdTs(m5dCs)usususgsc UUGC GUGU NM_000688.5_910- 910gsascsascsdTs(m5dCs)dTsdTsdTs 375 GACACUCUUUCUGGUC 645 CAAAGACCAGAAAGAG915 929_aso (m5dCs)dTsdGsdGsdTscsusususg UUUG UGUC NM_000688.5_911- 911asgsascsas(m5dCs)dTs(m5dCs)dTsdTs 376 AGACACUCUUUCUGG 646AAAGACCAGAAAGAGU 916 930_aso dTs(m5dCs)dTsdGsdGsuscsususu UCUUU GUCUNM_000688.5_912- 912 gsasgsascsdAs(m5dCs)dTs(m5dCs)dT 377GAGACACUCUUUCUG 647 AAGACCAGAAAGAGUG 917 931_asosdTsdTs(m5dCs)dTsdGsgsuscsusu GUCUU UCUC NM_000688.5_913- 913usgsasgsas(m5dCs)dAs(m5dCs)dTs 378 UGAGACACUCUUUCU 648 AGACCAGAAAGAGUGU918 932_aso (m5dCs)dTsdTsdTs(m5dCs)dTsgsgsuscs GGUCU CUCA uNM_000688.5_914- 914 asusgsasgsdAs(m5dCs)dAs(m5dCs)dT 379AUGAGACACUCUUUC 649 GACCAGAAAGAGUGUC 919 933_asos(m5dCs)dTsdTsdTs(m5dCs)usgsgsus UGGUC UCAU c NM_000688.5_915- 915gsasusgsasdGsdAs(m5dCs)dAs(m5dCs) 380 GAUGAGACACUCUUU 650ACCAGAAAGAGUGUCU 920 934_aso dTs(m5dCs)dTsdTsdTscsusgsgsu CUGGU CAUCNM_000688.5_916- 916 asgsasusgsdAsdGsdAs(m5dCs)dAs 381 AGAUGAGACACUCUU651 CCAGAAAGAGUGUCUC 921 935_aso (m5dCs)dTs(m5dCs)dTsdTsuscsusgsg UCUGGAUCU NM_000688.5_917- 917 asasgsasusdGsdAsdGsdAs(m5dCs)dA 382AAGAUGAGACACUCU 652 CAGAAAGAGUGUCUCA 922 936_asos(m5dCs)dTs(m5dCs)dTsususcsusg UUCUG UCUU NM_000688.5_918- 918gsasasgsasdTsdGsdAsdGsdAs(m5dCs) 383 GAAGAUGAGACACUC 653AGAAAGAGUGUCUCAU 923 937_aso dAs(m5dCs)dTs(m5dCs)usususcsu UUUCU CUUCNM_000688.5_919- 919 asgsasasgsdAsdTsdGsdAsdGsdAs 384 AGAAGAUGAGACACU654 GAAAGAGUGUCUCAUC 924 938_aso (m5dCs)dAs(m5dCs)dTscsusususc CUUUCUUCU NM_000688.5_920- 920 asasgsasasdGsdAsdTsdGsdAsdGsdAs 385AAGAAGAUGAGACAC 655 AAAGAGUGUCUCAUCU 925 939_aso(m5dCs)dAs(m5dCs)uscsususu UCUUU UCUU NM_000688.5_921- 921gsasasgsasdAsdGsdAsdTsdGsdAsdGs 386 GAAGAAGAUGAGACA 656 AAGAGUGUCUCAUCUU926 940_aso dAs(m5dCs)dAscsuscsusu CUCUU CUUC NM_000688.5_922- 922usgsasasgsdAsdAsdGsdAsdTsdGsdAs 387 UGAAGAAGAUGAGAC 657 AGAGUGUCUCAUCUUC927 941_aso dGsdAs(m5dCs)ascsuscsu ACUCU UUCA NM_000688.5_923- 923ususgsasasdGsdAsdAsdGsdAsdTsdGs 388 UUGAAGAAGAUGAGA 658 GAGUGUCUCAUCUUCU928 942_aso dAsdGsdAscsascsusc CACUC UCAA NM_000688.5_924- 924csususgsasdAsdGsdAsdAsdGsdAsdTs 389 CUUGAAGAAGAUGAG 659 AGUGUCUCAUCUUCUU929 943_aso dGsdAsdGsascsascsu ACACU CAAG NM_000688.5_925- 925uscsususgsdAsdAsdGsdAsdAsdGsdAs 390 UCUUGAAGAAGAUGA 660 GUGUCUCAUCUUCUUC930 944_aso dTsdGsdAsgsascsasc GACAC AAGA NM_000688.5_926- 926asuscsususdGsdAsdAsdGsdAsdAsdGs 391 AUCUUGAAGAAGAUG 661 UGUCUCAUCUUCUUCA931 945_aso dAsdTsdGsasgsascsa AGACA AGAU NM_000688.5_927- 927usasuscsusdTsdGsdAsdAsdGsdAsdAs 392 UAUCUUGAAGAAGAU 662 GUCUCAUCUUCUUCAA932 946_aso dGsdAsdTsgsasgsasc GAGAC GAUA NM_000688.5_928- 928ususasuscsdTsdTsdGsdAsdAsdGsdAs 393 UUAUCUUGAAGAAGA 663 UCUCAUCUUCUUCAAG933 947_aso dAsdGsdAsusgsasgsa UGAGA AUAA NM_000688.5_929- 929gsususasus(m5dCs)dTsdTsdGsdAsdAs 394 GUUAUCUUGAAGAAG 664CUCAUCUUCUUCAAGA 934 948_aso dGsdAsdAsdGsasusgsasg AUGAG UAACNM_000688.5_930- 930 asgsususasdTs(m5dCs)dTsdTsdGsdAs 395AGUUAUCUUGAAGAA 665 UCAUCUUCUUCAAGAU 935 949_aso dAsdGsdAsdAsgsasusgsaGAUGA AACU NM_000688.5_931- 931 asasgsususdAsdTs(m5dCs)dTsdTsdGs 396AAGUUAUCUUGAAGA 666 CAUCUUCUUCAAGAUA 936 950_aso dAsdAsdGsdAsasgsasusgAGAUG ACUU NM_000688.5_940- 940 gsasusususdTsdGsdGs(m5dCs)dAsdA 397GAUUUUGGCAAGUUA 667 CAAGAUAACUUGCCAA 937 959_aso sdGsdTsdTsdAsuscsususgUCUUG AAUC NM_000688.5_950- 950 asgsusgsgsdAsdAsdAs(m5dCs)dAsdG 398AGUGGAAACAGAUUU 668 UGCCAAAAUCUGUUUC 938 969_aso sdAsdTsdTsdTsusgsgscsaUGGCA CACU NM_000688.5_960- 960 csasusascsdTsdGsdAsdAsdAsdAsdGs 399CAUACUGAAAAGUGG 669 UGUUUCCACUUUUCAG 939 979_aso dTsdGsdGsasasascsaAAACA UAUG NM_000688.5_970- 970 asasgsasasdAs(m5dCs)dGsdAsdTs 400AAGAAACGAUCAUAC 670 UUUCAGUAUGAUCGUU 940 989_aso(m5dCs)dAsdTsdAs(m5dCs)usgsasasa UGAAA UCUU NM_000688.5_980- 980usususususdTs(m5dCs)dTs(m5dCs)dA 401 UUUUUUCUCAAAGAA 671AUCGUUUCUUUGAGAA 941 999_aso sdAsdAsdGsdAsdAsascsgsasu ACGAU AAAANM_000688.5_990- 990 uscsuscsasdTs(m5dCs)dAsdAsdTsdTs 402UCUCAUCAAUUUUUU 672 UGAGAAAAAAAUUGAU 942 1009_aso dTsdTsdTsdTsuscsuscsaUCUCA GAGA NM_000688.5_1000- 1000 uscsasusus(m5dCs)dTsdTsdTsdTsdTs 403UCAUUCUUUUUCUCA 673 AUUGAUGAGAAAAAGA 943 1019_aso(m5dCs)dTs(m5dCs)dAsuscsasasu UCAAU AUGA NM_000688.5_1010- 1010asusasgsgsdTsdGsdTsdGsdGsdTs(m5dCs) 404 AUAGGUGUGGUCAUU 674AAAAGAAUGACCACAC 944 1029_aso dAsdTsdTscsusususu CUUUU CUAUNM_000688.5_1020- 1020 usasasasasdAs(m5dCs)dTs(m5dCs)dG 405UAAAAACUCGAUAGG 675 CCACACCUAUCGAGUU 945 1039_asosdAsdTsdAsdGsdGsusgsusgsg UGUGG UUUA NM_000688.5_1030- 1030ususcsascsdAsdGsdTsdTsdTsdTsdAsd 406 UUCACAGUUUUAAAA 676CGAGUUUUUAAAACUG 946 1049_aso AsdAsdAsascsuscsg ACUCG UGAANM_000688.5_1040- 1040 usgscsuscsdGs(m5dCs)(m5dCs)dGsdG 407UGCUCGCCGGUUCACA 677 AAACUGUGAACCGGCG 947 1059_asosdTsdTs(m5dCs)dAs(m5dCs)asgsusus GUUU AGCA u NM_000688.5_1050- 1050gsgsasasgsdAsdTsdGsdTsdGsdTsdGs 408 GGAAGAUGUGUGCUC 678 CCGGCGAGCACACAUC948 1069_aso (m5dCs)dTs(m5dCs)gscscsgsg GCCGG UUCC NM_000688.5_1060-1060 uscsusgscs(m5dCs)dAsdTsdGsdGsdGs 409 UCUGCCAUGGGGAAG 679CACAUCUUCCCCAUGG 949 1079_aso dGsdAsdAsdGsasusgsusg AUGUG CAGANM_000688.5_1070- 1070 usgsasasusdAsdGsdTs(m5dCs)dAsdTs 410UGAAUAGUCAUCUGC 680 CCAUGGCAGAUGACUA 950 1089_aso(m5dCs)dTsdGs(m5dCs)csasusgsg CAUGG UUCA NM_000688.5_1080- 1080usgsasgsgsdGsdAsdGsdTs(m5dCs)dTs 411 UGAGGGAGUCUGAAU 681UGACUAUUCAGACUCC 951 1099_aso dGsdAsdAsdTsasgsuscsa AGUCA CUCANM_000688.5_1090- 1090 usususususdGsdGsdTsdGsdAsdTsdGs 412UUUUUGGUGAUGAGG 682 GACUCCCUCAUCACCA 952 1109_aso dAsdGsdGsgsasgsuscGAGUC AAAA NM_000688.5_1100- 1100 usgsascsas(m5dCs)dTsdTsdGs(m5dCs) 413UGACACUUGCUUUUU 683 UCACCAAAAAGCAAGU 953 1119_asodTsdTsdTsdTsdTsgsgsusgsa GGUGA GUCA NM_000688.5_1110- 1110usgscsascs(m5dCs)dAsdGsdAs(m5dCs) 414 UGCACCAGACUGACAC 684GCAAGUGUCAGUCUGG 954 1129_aso dTsdGsdAs(m5dCs)dAscsususgsc UUGC UGCANM_000688.5_1120- 1120 usasgsuscsdAsdTsdTsdAs(m5dCs)dTs 415UAGUCAUUACUGCACC 685 GUCUGGUGCAGUAAUG 955 1139_asodGs(m5dCs)dAs(m5dCs)csasgsasc AGAC ACUA NM_000688.5_1130- 1130csasususcs(m5dCs)dTsdAsdGsdGsdTs 416 CAUUCCUAGGUAGUC 686GUAAUGACUACCUAGG 956 1149_aso dAsdGsdTs(m5dCs)asususasc AUUAC AAUGNM_000688.5_1140- 1140 gsgsusgsgs(m5dCs)dGsdAs(m5dCs)dT 417GGUGGCGACUCAUUCC 687 CCUAGGAAUGAGUCGC 957 1159_asos(m5dCs)dAsdTsdTs(m5dCs)csusasgs UAGG CACC g NM_000688.5_1150- 1150csascsascs(m5dCs)(m5dCs)dGsdTsdG 418 CACACCCGUGGGUGGC 688AGUCGCCACCCACGGG 958 1169_aso sdGsdGsdTsdGsdGscsgsascsu GACU UGUGNM_000688.5_1160- 1160 asascsusgs(m5dCs)(m5dCs)(m5dCs) 419AACUGCCCCACACACC 689 CACGGGUGUGUGGGGC 959 1179_aso(m5dCs)dAs(m5dCs)dAs(m5dCs)dAs CGUG AGUU (m5dCs)cscsgsusgNM_000688.5_1170- 1170 asasgsusgsdTs(m5dCs)(m5dCs)dAsdT 420AAGUGUCCAUAACUG 690 UGGGGCAGUUAUGGAC 960 1189_asosdAsdAs(m5dCs)dTsdGscscscscsa CCCCA ACUU NM_000688.5_1180- 1180usgsususgsdTsdTsdTs(m5dCs)dAsdAs 421 UGUUGUUUCAAAGUG 691AUGGACACUUUGAAAC 961 1199_aso dAsdGsdTsdGsuscscsasu UCCAU AACANM_000688.5_1190- 1190 cscscsasgs(m5dCs)dAs(m5dCs)(m5dCs) 422CCCAGCACCAUGUUGU 692 UGAAACAACAUGGUGC 962 1209_asodAsdTsdGsdTsdTsdGsusususcsa UUCA UGGG NM_000688.5_1200- 1200usascscsas(m5dCs)(m5dCs)dTsdGs 423 UACCACCUGCCCCAGC 693 UGGUGCUGGGGCAGGU963 1219_aso (m5dCs)(m5dCs)(m5dCs)(m5dCs)dAsdG ACCA GGUA scsascscsaNM_000688.5_1210- 1210 asusasususdTs(m5dCs)dTsdAsdGsdTs 424AUAUUUCUAGUACCA 694 GCAGGUGGUACUAGAA 964 1229_asodAs(m5dCs)(m5dCs)dAscscsusgsc CCUGC AUAU NM_000688.5_1220- 1220asgsususcs(m5dCs)dAsdGsdAsdAsdA 425 AGUUCCAGAAAUAUU 695 CUAGAAAUAUUUCUGG965 1239_aso sdTsdAsdTsdTsuscsusasg UCUAG AACU NM_000688.5_1230- 1230gsgsasasusdTsdTsdAs(m5dCs)dTsdAs 426 GGAAUUUACUAGUUC 696UUCUGGAACUAGUAAA 966 1249_aso dGsdTsdTs(m5dCs)csasgsasa CAGAA UUCCNM_000688.5_1240- 1240 asasgsuscs(m5dCs)dAs(m5dCs)dAsdT 427AAGUCCACAUGGAAU 697 AGUAAAUUCCAUGUGG 967 1259_asosdGsdGsdAsdAsdTsususascsu UUACU ACUU NM_000688.5_1250- 1250csuscscscsdGs(m5dCs)dTs(m5dCs)dT 428 CUCCCGCUCUAAGUCC 698AUGUGGACUUAGAGCG 968 1269_aso sdAsdAsdGsdTs(m5dCs)csascsasu ACAU GGAGNM_000688.5_1260- 1260 gsgsuscsusdGs(m5dCs)(m5dCs)dAsd 429GGUCUGCCAGCUCCCG 699 AGAGCGGGAGCUGGCA 969 1279_asoGs(m5dCs)dTs(m5dCs)(m5dCs)(m5d CUCU GACC Cs)gscsuscsu NM_000688.5_1270-1270 ususcscscsdAsdTsdGsdGsdAsdGsdGs 430 UUCCCAUGGAGGUCU 700CUGGCAGACCUCCAUG 970 1289_aso dTs(m5dCs)dTsgscscsasg GCCAG GGAANM_000688.5_1280- 1280 usgscsgsgs(m5dCs)dAsdTs(m5dCs)dT 431UGCGGCAUCUUUCCCA 701 UCCAUGGGAAAGAUGC 971 1299_asosdTsdTs(m5dCs)(m5dCs)(m5dCs)asus UGGA CGCA gsgsa NM_000688.5_1290- 1290asasasascsdAsdAsdGsdAsdGsdTsdGs 432 AAAACAAGAGUGCGG 702 AGAUGCCGCACUCUUG972 1309_aso (m5dCs)dGsdGscsasuscsu CAUCU UUUU NM_000688.5_1300- 1300asasgscsas(m5dCs)dGsdAsdGsdGsdAs 433 AAGCACGAGGAAAAC 703CUCUUGUUUUCCUCGU 973 1319_aso dAsdAsdAs(m5dCs)asasgsasg AAGAG GCUUNM_000688.5_1310- 1310 asususgsgs(m5dCs)(m5dCs)dAs(m5dCs) 434AUUGGCCACAAAGCAC 704 CCUCGUGCUUUGUGGC 974 1329_asodAsdAsdAsdGs(m5dCs)dAscsgsasgs GAGG CAAU g NM_000688.5_1320- 1320gsgsgsususdGsdAsdGsdTs(m5dCs)dA 435 GGGUUGAGUCAUUGG 705 UGUGGCCAAUGACUCA975 1339_aso sdTsdTsdGsdGscscsascsa CCACA ACCC NM_000688.5_1330- 1330asgsgsgsusdGsdAsdAsdGsdAsdGsdGs 436 AGGGUGAAGAGGGUU 706 GACUCAACCCUCUUCA976 1349_aso dGsdTsdTsgsasgsusc GAGUC CCCU NM_000688.5_1340- 1340csasuscsusdTsdAsdGs(m5dCs)(m5dCs) 437 CAUCUUAGCCAGGGU 707UCUUCACCCUGGCUAA 977 1359_aso dAsdGsdGsdGsdTsgsasasgsa GAAGA GAUGNM_000688.5_1350- 1350 asgscscsusdGsdGs(m5dCs)dAsdTs 438AGCCUGGCAUCAUCUU 708 GGCUAAGAUGAUGCCA 978 1369_aso(m5dCs)dAsdTs(m5dCs)dTsusasgscsc AGCC GGCU NM_000688.5_1360- 1360usasasasus(m5dCs)dTs(m5dCs)dAs 439 UAAAUCUCACAGCCUG 709 AUGCCAGGCUGUGAGA979 1379_aso (m5dCs)dAsdGs(m5dCs)(m5dCs)dTsgsg GCAU UUUA scsasuNM_000688.5_1370- 1370 asgsasasus(m5dCs)dAsdGsdAsdGsdTs 440AGAAUCAGAGUAAAU 710 GUGAGAUUUACUCUGA 980 1389_aso dAsdAsdAsdTscsuscsascCUCAC UUCU NM_000688.5_1380- 1380 csasusgsgsdTsdTs(m5dCs)(m5dCs) 441CAUGGUUCCCAGAAUC 711 CUCUGAUUCUGGGAAC 981 1399_aso(m5dCs)dAsdGsdAsdAsdTscsasgsasg AGAG CAUG NM_000688.5_1390- 1390asuscsasusdGsdGsdAsdGsdGs(m5dCs) 442 AUCAUGGAGGCAUGG 712GGGAACCAUGCCUCCA 982 1409_aso dAsdTsdGsdGsususcscsc UUCCC UGAUNM_000688.5_1400- 1400 asasuscscs(m5dCs)dTsdTsdGsdGsdAs 443AAUCCCUUGGAUCAU 713 CCUCCAUGAUCCAAGG 983 1419_asodTs(m5dCs)dAsdTsgsgsasgsg GGAGG GAUU NM_000688.5_1410- 1410gsgscsusgsdTsdTsdTs(m5dCs)dGsdAs 444 GGCUGUUUCGAAUCCC 714CCAAGGGAUUCGAAAC 984 1429_aso dAsdTs(m5dCs)(m5dCs)csususgsg UUGG AGCCNM_000688.5_1420- 1420 usususgsgs(m5dCs)dAs(m5dCs)dTs 445UUUGGCACUCGGCUG 715 CGAAACAGCCGAGUGC 985 1439_aso(m5dCs)dGsdGs(m5dCs)dTsdGsusususcs UUUCG CAAA g NM_000688.5_1430- 1430gsasasgsasdTsdGsdTsdAs(m5dCs)dTs 446 GAAGAUGUACUUUGG 716GAGUGCCAAAGUACAU 986 1449_aso dTsdTsdGsdGscsascsusc CACUC CUUCNM_000688.5_1440- 1440 csasususgsdTsdGsdGs(m5dCs)dGsdGs 447CAUUGUGGCGGAAGA 717 GUACAUCUUCCGCCAC 987 1459_aso dAsdAsdGsdAsusgsusascUGUAC AAUG NM_000688.5_1450- 1450 usgsgscsusdGsdAs(m5dCs)dAsdTs 448UGGCUGACAUCAUUG 718 CGCCACAAUGAUGUCA 988 1469_aso(m5dCs)dAsdTsdTsdGsusgsgscsg UGGCG GCCA NM_000688.5_1460- 1460ususcsuscsdTsdGsdAsdGsdGsdTsdGs 449 UUCUCUGAGGUGGCU 719 AUGUCAGCCACCUCAG989 1479_aso dGs(m5dCs)dTsgsascsasu GACAU AGAA NM_000688.5_1470- 1470usususgscsdAsdGs(m5dCs)dAsdGsdT 450 UUUGCAGCAGUUCUC 720 CCUCAGAGAACUGCUG990 1489_aso sdTs(m5dCs)dTs(m5dCs)usgsasgsg UGAGG CAAA NM_000688.5_1480-1480 gsgsgsuscsdAsdGsdAsdTs(m5dCs)dTs 451 GGGUCAGAUCUUUGC 721CUGCUGCAAAGAUCUG 991 1499_aso dTsdTsdGs(m5dCs)asgscsasg AGCAG ACCCNM_000688.5_1490- 1490 gsgsgsgsas(m5dCs)dTsdGsdAsdGsdG 452GGGGACUGAGGGGUC 722 GAUCUGACCCCUCAGU 992 1509_asosdGsdGsdTs(m5dCs)asgsasusc AGAUC CCCC NM_000688.5_1500- 1500cscsascsasdAsdTs(m5dCs)dTsdTsdGs 453 CCACAAUCUUGGGGAC 723CUCAGUCCCCAAGAUU 993 1519_aso dGsdGsdGsdAscsusgsasg UGAG GUGGNM_000688.5_1510- 1510 gsusususcsdAsdAsdAsdTsdGs(m5dCs) 454GUUUCAAAUGCCACA 724 AAGAUUGUGGCAUUUG 994 1529_aso(m5dCs)dAs(m5dCs)dAsasuscsusu AUCUU AAAC NM_000688.5_1520- 1520usgsasasusdGsdGsdAs(m5dCs)dAsdG 455 UGAAUGGACAGUUUC 725 CAUUUGAAACUGUCCA995 1539_aso sdTsdTsdTs(m5dCs)asasasusg AAAUG UUCA NM_000688.5_1530-1530 cscscscsasdTs(m5dCs)(m5dCs)dAsdTs 456 CCCCAUCCAUUGAAUG 726UGUCCAUUCAAUGGAU 996 1549_aso dTsdGsdAsdAsdTsgsgsascsa GACA GGGGNM_000688.5_1540- 1540 gsgsgscsas(m5dCs)dAs(m5dCs)(m5dCs) 457GGGCACACCGCCCCAU 727 AUGGAUGGGGCGGUGU 997 1559_asodGs(m5dCs)(m5dCs)(m5dCs)(m5dCs) CCAU GCCC dAsuscscsasu NM_000688.5_1550-1550 csuscsusus(m5dCs)(m5dCs)dAsdGsdT 458 CUCUUCCAGUGGGCAC 728CGGUGUGCCCACUGGA 998 1569_aso sdGsdGsdGs(m5dCs)dAscsascscsg ACCG AGAGNM_000688.5_1560- 1560 csasuscsas(m5dCs)dAs(m5dCs)dAsdG 459CAUCACACAGCUCUUC 729 ACUGGAAGAGCUGUGU 999 1579_asos(m5dCs)dTs(m5dCs)dTsdTscscsasgsu CAGU GAUG NM_000688.5_1570- 1570uscsasusgsdGsdGs(m5dCs)(m5dCs)dA 460 UCAUGGGCCACAUCAC 730CUGUGUGAUGUGGCCC 1000 1589_aso s(m5dCs)dAsdTs(m5dCs)dAscsascsas ACAGAUGA g NM_000688.5_1580- 1580 usgscsuscs(m5dCs)dAsdAsdAs(m5dCs) 461UGCUCCAAACUCAUGG 731 UGGCCCAUGAGUUUGG 1001 1599_asodTs(m5dCs)dAsdTsdGsgsgscscsa GCCA AGCA NM_000688.5_1590- 1590csgsasasgsdGsdTsdGsdAsdTsdTsdGs 462 CGAAGGUGAUUGCUC 732 GUUUGGAGCAAUCACC1002 1609_aso (m5dCs)dTs(m5dCs)csasasasc CAAAC UUCG NM_000688.5_1600-1600 ascscsuscsdAsdTs(m5dCs)(m5dCs)dA 463 ACCUCAUCCACGAAGG 733AUCACCUUCGUGGAUG 1003 1619_aso s(m5dCs)dGsdAsdAsdGsgsusgsasu UGAU AGGUNM_000688.5_1610- 1610 csascsusgs(m5dCs)dGsdTsdGsdGsdAs 464CACUGCGUGGACCUCA 734 UGGAUGAGGUCCACGC 1004 1629_aso(m5dCs)(m5dCs)dTs(m5dCs)asuscscs UCCA AGUG a NM_000688.5_1620- 1620csasusasasdAsdGs(m5dCs)(m5dCs) 465 CAUAAAGCCCCACUGC 735 CCACGCAGUGGGGCUU1005 1639_aso (m5dCs)(m5dCs)dAs(m5dCs)dTsdGscsg GUGG UAUG susgsgNM_000688.5_1630- 1630 cscsuscsgsdAsdGs(m5dCs)(m5dCs) 466CCUCGAGCCCCAUAAA 736 GGGCUUUAUGGGGCUC 1006 1649_aso(m5dCs)(m5dCs)dAsdTsdAsdAsasgscscs GCCC GAGG c NM_000688.5_1640- 1640asasuscscs(m5dCs)dTs(m5dCs)(m5dCs) 467 AAUCCCUCCGCCUCGA 737GGGCUCGAGGCGGAGG 1007 1659_aso dGs(m5dCs)(m5dCs)dTs(m5dCs)dG GCCC GAUUsasgscscsc NM_000688.5_1650- 1650 cscscsgsasdTs(m5dCs)(m5dCs)(m5dCs) 468CCCGAUCCCCAAUCCC 738 CGGAGGGAUUGGGGAU 1008 1669_aso(m5dCs)dAsdAsdTs(m5dCs)(m5dCs) UCCG CGGG csuscscsg NM_000688.5_1660-1660 asusgsascsdTs(m5dCs)(m5dCs)dAsdT 469 AUGACUCCAUCCCGAU 739GGGGAUCGGGAUGGAG 1009 1679_aso s(m5dCs)(m5dCs)(m5dCs)dGsdAsusc CCCC UCAUscscsc NM_000688.5_1670- 1670 csasusususdTsdTsdGsdGs(m5dCs)dAs 470CAUUUUUGGCAUGAC 740 AUGGAGUCAUGCCAAA 1010 1689_asodTsdGsdAs(m5dCs)uscscsasu UCCAU AAUG NM_000688.5_1680- 1680asasasusgsdAsdTsdGsdTs(m5dCs) 471 AAAUGAUGUCCAUUU 741 GCCAAAAAUGGACAUC1011 1699_aso (m5dCs)dAsdTsdTsdTsususgsgsc UUGGC AUUU NM_000688.5_1690-1690 asgsusgsusdTs(m5dCs)(m5dCs)dAsdG 472 AGUGUUCCAGAAAUG 742GACAUCAUUUCUGGAA 1012 1709_aso sdAsdAsdAsdTsdGsasusgsusc AUGUC CACUNM_000688.5_1700- 1700 gsgscsususdTsdGs(m5dCs)(m5dCs)dA 473GGCUUUGCCAAGUGU 743 CUGGAACACUUGGCAA 1013 1719_asosdAsdGsdTsdGsdTsuscscsasg UCCAG AGCC NM_000688.5_1710- 1710csascsasas(m5dCs)(m5dCs)dAsdAsdA 474 CACAACCAAAGGCUUU 744UGGCAAAGCCUUUGGU 1014 1729_aso sdGsdGs(m5dCs)dTsdTsusgscscsa GCCA UGUGNM_000688.5_1720- 1720 usascscscsdTs(m5dCs)(m5dCs)dAsdA 475UACCCUCCAACACAAC 745 UUUGGUUGUGUUGGAG 1015 1739_asos(m5dCs)dAs(m5dCs)dAsdAscscsasas CAAA GGUA a NM_000688.5_1730- 1730gscsusgsgs(m5dCs)dGsdAsdTsdGsdTs 476 GCUGGCGAUGUACCCU 746UUGGAGGGUACAUCGC 1016 1749_aso dAs(m5dCs)(m5dCs)(m5dCs)uscscsas CCAACAGC a NM_000688.5_1740- 1740 gsasgsasas(m5dCs)dTs(m5dCs)dGsdT 477GAGAACUCGUGCUGG 747 CAUCGCCAGCACGAGU 1017 1759_asosdGs(m5dCs)dTsdGsdGscsgsasusg CGAUG UCUC NM_000688.5_1750- 1750gsusgsuscsdAsdAsdTs(m5dCs)dAsdG 478 GUGUCAAUCAGAGAA 748 ACGAGUUCUCUGAUUG1018 1769_aso sdAsdGsdAsdAscsuscsgsu CUCGU ACAC NM_000688.5_1760- 1760gsgsascscsdGsdTsdAs(m5dCs)dGsdGs 479 GGACCGUACGGUGUC 749UGAUUGACACCGUACG 1019 1779_aso dTsdGsdTs(m5dCs)asasuscsa AAUCA GUCCNM_000688.5_1770- 1770 csasgscsasdGs(m5dCs)dAsdTsdAsdGs 480CAGCAGCAUAGGACCG 750 CGUACGGUCCUAUGCU 1020 1789_asodGsdAs(m5dCs)(m5dCs)gsusascsg UACG GCUG NM_000688.5_1780- 1780asasgsasusdGsdAsdAsdGs(m5dCs) 481 AAGAUGAAGCCAGCA 751 UAUGCUGCUGGCUUCA1021 1799_aso (m5dCs)dAsdGs(m5dCs)dAsgscsasusa GCAUA UCUUNM_000688.5_1790- 1790 asgsasgsgsdTsdGsdGsdTsdGsdAsdAs 482AGAGGUGGUGAAGAU 752 GCUUCAUCUUCACCAC 1022 1809_aso dGsdAsdTsgsasasgscGAAGC CUCU NM_000688.5_1800- 1800 usgsgsgsusdGsdGs(m5dCs)dAsdGsdA 483UGGGUGGCAGAGAGG 753 CACCACCUCUCUGCCA 1023 1819_asosdGsdAsdGsdGsusgsgsusg UGGUG CCCA NM_000688.5_1810- 1810gscscsasgs(m5dCs)dAsdGs(m5dCs)dA 484 GCCAGCAGCAUGGGU 754CUGCCACCCAUGCUGC 1024 1829_aso sdTsdGsdGsdGsdTsgsgscsasg GGCAG UGGCNM_000688.5_1820- 1820 csasgsgsgs(m5dCs)dTs(m5dCs)(m5dCs) 485CAGGGCUCCAGCCAGC 755 UGCUGCUGGCUGGAGC 1025 1839_asodAsdGs(m5dCs)(m5dCs)dAsdGscsa AGCA CCUG sgscsa NM_000688.5_1830- 1830gscsascsasdGsdAs(m5dCs)dTs(m5dCs) 486 GCACAGACUCCAGGGC 756UGGAGCCCUGGAGUCU 1026 1849_aso (m5dCs)dAsdGsdGsdGscsuscscsa UCCA GUGCNM_000688.5_1840- 1840 ususcsasgsdGsdAsdTs(m5dCs)(m5dCs) 487UUCAGGAUCCGCACAG 757 GAGUCUGUGCGGAUCC 1027 1859_asodGs(m5dCs)dAs(m5dCs)dAsgsascsus ACUC UGAA c NM_000688.5_1850- 1850csuscsasgs(m5dCs)dGs(m5dCs)dTs 488 CUCAGCGCUCUUCAGG 758 GGAUCCUGAAGAGCGC1028 1869_aso (m5dCs)dTsdTs(m5dCs)dAsdGsgsasuscs AUCC UGAG cNM_000688.5_1860- 1860 gscsascscs(m5dCs)dGsdTs(m5dCs) 489GCACCCGUCCCUCAGC 759 GAGCGCUGAGGGACGG 1029 1879_aso(m5dCs)(m5dCs)dTs(m5dCs)dAsdGscsg GCUC GUGC scsusc NM_000688.5_1870-1870 usgsgscsgsdGs(m5dCs)dGsdAsdAsdG 490 UGGCGGCGAAGCACCC 760GGACGGGUGCUUCGCC 1030 1889_aso s(m5dCs)dAs(m5dCs)(m5dCs)csgsusc GUCCGCCA sc NM_000688.5 1880- 1880 gscsgscsusdGsdGsdTsdGs(m5dCs)dTs 491GCGCUGGUGCUGGCG 761 UUCGCCGCCAGCACCA 1031 1899_asodGsdGs(m5dCs)dGsgscsgsasa GCGAA GCGC NM_000688.5_1890- 1890gsusususgsdAs(m5dCs)dGsdTsdTsdGs 492 GUUUGACGUUGCGCU 762GCACCAGCGCAACGUC 1032 1909_aso (m5dCs)dGs(m5dCs)dTsgsgsusgsc GGUGC AAACNM_000688.5_1900- 1900 usgsuscsus(m5dCs)dAsdTsdGsdAsdG 493UGUCUCAUGAGUUUG 763 AACGUCAAACUCAUGA 1033 1919_asosdTsdTsdTsdGsascsgsusu ACGUU GACA NM_000688.5_1910- 1910csasususasdGs(m5dCs)dAsdTs(m5dCs) 494 CAUUAGCAUCUGUCUC 764UCAUGAGACAGAUGCU 1034 1929_aso dTsdGsdTs(m5dCs)dTscsasusgsa AUGA AAUGNM_000688.5_1920- 1920 gsgscscsgsdGs(m5dCs)dAsdTs(m5dCs) 495GGCCGGCAUCCAUUAG 765 GAUGCUAAUGGAUGCC 1035 1939_aso(m5dCs)dAsdTsdTsdAsgscsasusc CAUC GGCC NM_000688.5_1930- 1930ascsasascsdAsdGsdGsdGsdAsdGsdGs 496 ACAACAGGGAGGCCG 766 GAUGCCGGCCUCCCUG1036 1949_aso (m5dCs)(m5dCs)dGsgscsasusc GCAUC UUGU NM_000688.5_1940-1940 gsgsgsgscsdAsdGsdTsdGsdGsdAs 497 GGGGCAGUGGACAAC 767UCCCUGUUGUCCACUG 1037 1959_aso (m5dCs)dAsdAs(m5dCs)asgsgsgsa AGGGA CCCCNM_000688.5_1950- 1950 usgsasusgsdTsdGsdGs(m5dCs)dTsdGs 498UGAUGUGGCUGGGGC 768 CCACUGCCCCAGCCAC 1038 1969_asodGsdGsdGs(m5dCs)asgsusgsg AGUGG AUCA NM_000688.5_1960- 1960csgscsascsdAsdGsdGsdGsdAsdTsdGs 499 CGCACAGGGAUGAUG 769 AGCCACAUCAUCCCUG1039 1979_aso dAsdTsdGsusgsgscsu UGGCU UGCG NM_000688.5_1970- 1970asuscsusgs(m5dCs)dAsdAs(m5dCs) 500 AUCUGCAACCCGCACA 770 UCCCUGUGCGGGUUGC1040 1989_aso (m5dCs)(m5dCs)dGs(m5dCs)dAs(m5dCs) GGGA AGAU asgsgsgsaNM_000688.5_1980- 1980 ususususasdGs(m5dCs)dAsdGs(m5dCs) 501UUUUAGCAGCAUCUG 771 GGUUGCAGAUGCUGCU 1041 1999_asodAsdTs(m5dCs)dTsdGscsasascsc CAACC AAAA NM_000688.5_1990- 1990ascsususcsdTsdGsdTsdGsdTsdTsdTsd 502 ACUUCUGUGUUUUUA 772GCUGCUAAAAACACAG 1042 2009_aso TsdTsdAsgscsasgsc GCAGC AAGUNM_000688.5_2000- 2000 ususcsasus(m5dCs)dAs(m5dCs)dAsdG 503UUCAUCACAGACUUCU 773 ACACAGAAGUCUGUGA 1043 2019_asosdAs(m5dCs)dTsdTs(m5dCs)usgsusgs GUGU UGAA u NM_000688.5_2010- 2010usgscsuscsdAsdTsdTsdAsdGsdTsdTs 504 UGCUCAUUAGUUCAU 774 CUGUGAUGAACUAAUG1044 2029_aso (m5dCs)dAsdTscsascsasg CACAG AGCA NM_000688.5_2020- 2020asusgsususdAsdTsdGsdTs(m5dCs)dTs 505 AUGUUAUGUCUGCUC 775CUAAUGAGCAGACAUA 1045 2039_aso dGs(m5dCs)dTs(m5dCs)asususasg AUUAG ACAUNM_000688.5_2030- 2030 ususgscsas(m5dCs)dGsdTsdAsdGsdAs 506UUGCACGUAGAUGUU 776 GACAUAACAUCUACGU 1046 2049_aso dTsdGsdTsdTsasusgsuscAUGUC GCAA NM_000688.5_2040- 2040 asasususgsdAsdTsdTsdGs(m5dCs)dTs 507AAUUGAUUGCUUGCA 777 CUACGUGCAAGCAAUC 1047 2059_asodTsdGs(m5dCs)dAscsgsusasg CGUAG AAUU NM_000688.5_2050- 2050ascscsgsusdAsdGsdGsdGsdTsdAsdAs 508 ACCGUAGGGUAAUUG 778 GCAAUCAAUUACCCUA1048 2069_aso dTsdTsdGsasususgsc AUUGC CGGU NM_000688.5_2060- 2060uscscscscsdGsdGsdGsdGs(m5dCs)dAs 509 UCCCCGGGGCACCGUA 779ACCCUACGGUGCCCCG 1049 2079_aso (m5dCs)(m5dCs)dGsdTsasgsgsgsu GGGU GGGANM_000688.5_2070- 2070 gsgsasgscsdTs(m5dCs)dTsdTs(m5dCs) 510GGAGCUCUUCUCCCCG 780 GCCCCGGGGAGAAGAG 1050 2089_asodTs(m5dCs)(m5dCs)(m5dCs)(m5dCs) GGGC CUCC gsgsgsgsc NM_000688.5_2080-2080 gscsasasus(m5dCs)(m5dCs)dGsdTsdA 511 GCAAUCCGUAGGAGC 781GAAGAGCUCCUACGGA 1051 2099_aso sdGsdGsdAsdGs(m5dCs)uscsususc UCUUC UUGCNM_000688.5_2090- 2090 asgsgsgsgsdTsdGsdGsdGsdGsdGs 512 AGGGGUGGGGGCAAU782 UACGGAUUGCCCCCAC 1052 2109_aso (m5dCs)dAsdAsdTscscsgsusa CCGUA CCCUNM_000688.5_2100- 2100 gsusgsusgsdTsdGsdGsdTsdGsdAsdGs 513GUGUGUGGUGAGGGG 783 CCCCACCCCUCACCACA 1053 2119_aso dGsdGsdGsusgsgsgsgUGGGG CAC NM_000688.5_2110- 2110 asuscsasus(m5dCs)dTsdGsdGsdGsdGs 514AUCAUCUGGGGUGUG 784 CACCACACACCCCAGA 1054 2129_aso dTsdGsdTsdGsusgsgsusgUGGUG UGAU NM_000688.5_2120- 2120 gsasasgsusdAsdGsdTsdTs(m5dCs)dAs 515GAAGUAGUUCAUCAU 785 CCCAGAUGAUGAACUA 1055 2139_asodTs(m5dCs)dAsdTscsusgsgsg CUGGG CUUC NM_000688.5_2130- 2130gsasususcsdTs(m5dCs)dAsdAsdGsdGs 516 GAUUCUCAAGGAAGU 786GAACUACUUCCUUGAG 1056 2149_aso dAsdAsdGsdTsasgsususc AGUUC AAUCNM_000688.5_2140- 2140 gsusgsascsdTsdAsdGs(m5dCs)dAsdGs 517GUGACUAGCAGAUUC 787 CUUGAGAAUCUGCUAG 1057 2159_asodAsdTsdTs(m5dCs)uscsasasg UCAAG UCAC NM_000688.5_2150- 2150ususgscsusdTs(m5dCs)(m5dCs)dAsdT 518 UUGCUUCCAUGUGAC 788UGCUAGUCACAUGGAA 1058 2169_aso sdGsdTsdGsdAs(m5dCs)usasgscsa UAGCA GCAANM_000688.5_2160- 2160 cscsasgscs(m5dCs)(m5dCs)(m5dCs)d 519CCAGCCCCACUUGCUU 789 AUGGAAGCAAGUGGGG 1059 2179_asoAs(m5dCs)dTsdTsdGs(m5dCs)dTsusc CCAU CUGG scsasu NM_000688.5_2170- 2170gsgscsusus(m5dCs)dAsdGsdTsdTs(m5 520 GGCUUCAGUUCCAGCC 790GUGGGGCUGGAACUGA 1060 2189_aso dCs)(m5dCs)dAsdGs(m5dCs)cscscsas CCACAGCC c NM_000688.5_2180- 2180 usgsasgsgsdAsdAsdTsdGsdAsdGsdGs 521UGAGGAAUGAGGCUU 791 AACUGAAGCCUCAUUC 1061 2199_aso(m5dCs)dTsdTscsasgsusu CAGUU CUCA NM_000688.5_2190- 2190usgscsascsdTs(m5dCs)dAsdGs(m5dCs) 522 UGCACUCAGCUGAGG 792UCAUUCCUCAGCUGAG 1062 2209_aso dTsdGsdAsdGsdGsasasusgsa AAUGA UGCANM_000688.5_2200- 2200 csusgscsasdGsdAsdAsdGsdTsdTsdGs 523CUGCAGAAGUUGCAC 793 GCUGAGUGCAACUUCU 1063 2219_aso(m5dCs)dAs(m5dCs)uscsasgsc UCAGC GCAG NM_000688.5_2210- 2210csasgsusgsdGs(m5dCs)(m5dCs)dTs 524 CAGUGGCCUCCUGCAG 794 ACUUCUGCAGGAGGCC1064 2229_aso (m5dCs)(m5dCs)dTsdGs(m5dCs)dAsgsa AAGU ACUG sasgsuNM_000688.5_2220- 2220 csususcsasdAsdAsdAsdTsdGs(m5dCs) 525CUUCAAAAUGCAGUG 795 GAGGCCACUGCAUUUU 1065 2239_aso dAsdGsdTsdGsgscscsuscGCCUC GAAG NM_000688.5_2230- 2230 uscsascsus(m5dCs)dAsdTs(m5dCs)dA 526UCACUCAUCACUUCAA 796 CAUUUUGAAGUGAUGA 1066 2249_asos(m5dCs)dTsdTs(m5dCs)dAsasasasus AAUG GUGA g NM_000688.5_2240- 2240csususcsus(m5dCs)dTs(m5dCs)dTsdT 527 CUUCUCUCUUUCACUC 797UGAUGAGUGAAAGAGA 1067 2259_aso sdTs(m5dCs)dAs(m5dCs)dTscsasuscs AUCAGAAG a NM_000688.5_2250- 2250 asgsasasasdTsdAsdGsdGsdAs(m5dCs) 528AGAAAUAGGACUUCU 798 AAGAGAGAAGUCCUAU 1068 2269_asodTsdTs(m5dCs)dTscsuscsusu CUCUU UUCU NM_000688.5_2260- 2260csuscsasasdGs(m5dCs)(m5dCs)dTsdG 529 CUCAAGCCUGAGAAA 799UCCUAUUUCUCAGGCU 1069 2279_aso sdAsdGsdAsdAsdAsusasgsgsa UAGGA UGAGNM_000688.5_2270- 2270 usascscsasdAs(m5dCs)dTsdTsdGs 530UACCAACUUGCUCAAG 800 CAGGCUUGAGCAAGUU 1070 2289_aso(m5dCs)dTs(m5dCs)dAsdAsgscscsusg CCUG GGUA NM_000688.5_2280- 2280cscsusgsasdGs(m5dCs)dAsdGsdAsdTs 531 CCUGAGCAGAUACCAA 801CAAGUUGGUAUCUGCU 1071 2299_aso dAs(m5dCs)(m5dCs)dAsascsususg CUUG CAGGNM_000688.5_2290- 2290 csasusgscsdTs(m5dCs)dAsdGsdGs 532CAUGCUCAGGCCUGAG 802 UCUGCUCAGGCCUGAG 1072 2309_aso(m5dCs)(m5dCs)dTsdGsdAsgscsasgsa CAGA CAUG NM_000688.5_2300- 2300usasasususdGsdAsdGsdGsdTs(m5dCs) 533 UAAUUGAGGUCAUGC 803CCUGAGCAUGACCUCA 1073 2319_aso dAsdTsdGs(m5dCs)uscsasgsg UCAGG AUUANM_000688.5_2310- 2310 ususasasgsdTsdGsdAsdAsdAsdTsdAs 534UUAAGUGAAAUAAUU 804 ACCUCAAUUAUUUCAC 1074 2329_aso dAsdTsdTsgsasgsgsuGAGGU UUAA NM_000688.5_2320- 2320 usgsgscscsdTsdGsdGsdGsdGsdTsdTsd 535UGGCCUGGGGUUAAG 805 UUUCACUUAACCCCAG 1075 2339_aso AsdAsdGsusgsasasaUGAAA GCCA NM_000688.5_2330- 2330 gsasusasusdGsdAsdTsdAsdAsdTsdGs 536GAUAUGAUAAUGGCC 806 CCCCAGGCCAUUAUCA 1076 2349_asodGs(m5dCs)(m5dCs)usgsgsgsg UGGGG UAUC NM_000688.5_2340- 2340asgsascscsdAsdTs(m5dCs)dTsdGsdGs 537 AGACCAUCUGGAUAU 807UUAUCAUAUCCAGAUG 1077 2359_aso dAsdTsdAsdTsgsasusasa GAUAA GUCUNM_000688.5_2350- 2350 ascsasascsdTs(m5dCs)dTsdGsdAsdAs 538ACAACUCUGAAGACCA 808 CAGAUGGUCUUCAGAG 1078 2369_asodGsdAs(m5dCs)(m5dCs)asuscsusg UCUG UUGU NM_000688.5_2360- 2360ascsasusasdTsdAsdAsdAsdGsdAs 539 ACAUAUAAAGACAAC 809 UCAGAGUUGUCUUUAU1079 2379_aso (m5dCs)dAsdAs(m5dCs)uscsusgsa UCUGA AUGU NM_000688.5_2370-2370 asascsususdAsdAsdTsdTs(m5dCs)dAs 540 AACUUAAUUCACAUA 810CUUUAUAUGUGAAUUA 1080 2389_aso (m5dCs)dAsdTsdAsusasasasg UAAAG AGUUNM_000688.5_2380- 2380 asasusususdAsdAsdTsdAsdTsdAsdAs 541AAUUUAAUAUAACUU 811 GAAUUAAGUUAUAUUA 1081 2399_aso(m5dCs)dTsdTsasasususc AAUUC AAUU NM_000688.5_2390- 2390usasusasgsdAsdTsdTsdAsdAsdAsdAs 542 UAUAGAUUAAAAUUU 812 AUAUUAAAUUUUAAUC1082 2409_aso dTsdTsdTsasasusasu AAUAU UAUA NM_000688.5_2400- 2400asusgsususdTsdTsdTsdAs(m5dCs)dTs 543 AUGUUUUUACUAUAG 813UUAAUCUAUAGUAAAA 1083 2419_aso dAsdTsdAsdGsasususasa AUUAA ACAUNM_000688.5_2410- 2410 ususcscsasdGsdGsdAs(m5dCs)dTsdAs 544UUCCAGGACUAUGUU 814 GUAAAAACAUAGUCCU 1084 2429_aso dTsdGsdTsdTsusususascUUUAC GGAA NM_000688.5_2420- 2420 asasgsasasdTsdTsdTsdAsdTsdTsdTs 545AAGAAUUUAUUUCCA 815 AGUCCUGGAAAUAAAU 1085 2439_aso(m5dCs)(m5dCs)dAsgsgsascsu GGACU UCUU NM_000688.5_2430- 2430cscsasususdTsdAsdAsdGs(m5dCs)dAs 546 CCAUUUAAGCAAGAA 816AUAAAUUCUUGCUUAA 1086 2449_aso dAsdGsdAsdAsusususasu UUUAU AUGG

Example 2. In Vitro Screening

In vitro screening of the antisense polynucleotides was performed bytransfecting Hep3B cells with a single 5 nM dose of an antisensepolynucleotide using methods well known in the art.

Briefly, a single 5 nM dose screen of each of 270 ALAS1 oligos wasperformed in Hep3B cells by seeding about 15,000 cells per well in 96well plates. Each oligo was transfected in quadruplicate with 0.5 μlLipofectamine 2000/well. Transfections were harvested 24 hours afterseeding/transfection. Transfection of an Aha1 LNA gapmer as a controltransfection, and mock transfections were performed in quadruplicate oneach plate. Mean values of ALAS1/GAPDH from Aha1-LNA transfection wasset as 100% ALAS1 expression, which is the reference for all other meanvalues shown in Table 5. At the same time, the AhaI LNA also served as atransfection control on each plate.

The complete screen was performed in two transfection “sessions”.Overall, transfection efficiency with an Aha1-oligo was between ˜90-95%at 5 nM. All ALAS1 oligos were less efficient than the Aha1-LNA at thesame concentration, the best producing a KD of ˜70%.

Table 5 shows the results of a single dose transfection screen in cellstransfected with the indicated antisense polynucleotide.

TABLE 5 Corrected transfection meanval % efficiency (w/o correction) sd% (tfe) X10361K2 97 7 96 X10362K2 86 5 85 X10363K2 95 4 94 X10364K2 81 780 X10365K2 93 4 92 X10366K2 92 9 91 X10367K2 79 9 78 X10368K2 77 6 76X10369K2 91 9 90 X10370K2 87 6 86 X10371K2 68 5 67 X10372K2 85 12 84X10373K2 89 7 88 X10374K2 87 5 86 X10375K2 90 11 89 X10376K2 90 7 89X10377K2 94 8 93 X10378K2 54 4 53 X10379K2 81 3 80 X10380K2 82 6 81X10381K2 91 4 89 X10382K2 92 2 90 X10383K2 101 5 100 X10384K2 99 11 97X10385K2 97 6 95 X10386K2 95 3 94 X10387K2 87 4 85 X10388K2 91 9 90X10389K2 75 5 74 X10390K2 70 3 68 X10391K2 85 18 84 X10392K2 88 8 86X10393K2 92 4 90 X10394K2 85 14 83 X10395K2 92 7 91 X10396K2 96 4 95X10397K2 85 3 83 X10398K2 73 4 71 X10399K2 90 5 88 X10400K2 94 6 92X10401K2 86 9 86 X10402K2 75 12 75 X10403K2 64 10 64 X10404K2 97 12 97X10405K2 96 9 96 X10406K2 111 13 111 X10407K2 90 12 90 X10408K2 116 19116 X10409K2 106 16 106 X10410K2 107 12 107 X10411K2 59 6 59 X10412K2 657 65 X10413K2 85 13 85 X10414K2 86 10 86 X10415K2 90 9 90 X10416K2 63 363 X10417K2 91 7 91 X10418K2 73 3 73 X10419K2 80 7 80 X10420K2 91 7 91X10421K2 68 8 67 X10422K2 60 4 59 X10423K2 64 5 63 X10424K2 80 8 79X10425K2 88 2 87 X10426K2 75 6 74 X10427K2 93 6 92 X10428K2 94 8 93X10429K2 92 5 91 X10430K2 71 7 70 X10431K2 67 14 66 X10432K2 59 4 59X10433K2 74 9 73 X10434K2 64 6 63 X10435K2 74 8 73 X10436K2 91 19 90X10437K2 92 7 91 X10438K2 88 10 87 X10439K2 108 9 107 X10440K2 101 8 100X10441K2 88 7 88 X10442K2 57 1 56 X10443K2 78 5 77 X10444K2 81 3 81X10445K2 61 7 60 X10446K2 71 6 71 X10447K2 69 4 68 X10448K2 102 5 101X10449K2 73 4 73 X10450K2 65 3 65 X10451K2 66 5 66 X10452K2 73 3 73X10453K2 75 5 75 X10454K2 96 8 96 X10455K2 92 4 91 X10456K2 79 5 79X10457K1 70 2 70 X10458K1 56 4 55 X10459K1 61 5 60 X10460K1 76 5 75X10461K1 97 3 94 X10462K1 98 4 95 X10463K1 93 15 90 X10464K1 95 10 92X10465K1 86 12 83 X10466K1 100 6 97 X10467K1 97 8 95 X10468K1 98 4 95X10469K1 90 4 87 X10470K1 99 2 96 X10471K1 110 4 107 X10472K1 122 2 119X10473K1 117 9 114 X10474K1 119 7 116 X10475K1 116 6 113 X10476K1 111 11108 X10477K1 108 8 105 X10478K1 108 8 106 X10479K1 108 10 105 X10480K1105 6 102 X10481K1 77 3 75 X10482K1 83 7 81 X10483K1 98 5 95 X10484K1102 10 99 X10485K1 107 11 104 X10486K1 111 12 109 X10487K1 112 8 109X10488K1 71 4 69 X10489K1 81 8 79 X10490K1 106 9 103 X10491K1 73 8 71X10492K1 56 4 53 X10493K1 88 9 85 X10494K1 68 8 65 X10495K1 85 9 82X10496K1 94 13 91 X10497K1 89 8 86 X10498K1 67 3 65 X10499K1 65 4 63X10500K1 102 11 100 X10501K1 56 9 53 X10502K1 78 6 75 X10503K1 68 7 65X10504K1 58 6 55 X10505K2 88 4 85 X10506K2 70 6 67 X10507K2 52 8 49X10508K2 89 8 86 X10509K2 97 5 94 X10510K2 88 8 85 X10511K2 70 14 67X10512K2 65 14 62 X10513K2 47 11 44 X10514K2 64 11 61 X10515K2 50 9 47X10516K1 77 11 74 X10517K2 73 5 70 X10518K2 42 6 39 X10519K2 33 7 30X10520K2 94 10 91 X10521K2 126 7 125 X10522K2 102 9 100 X10523K2 89 9 88X10524K2 113 4 112 X10525K2 67 3 66 X10526K2 73 5 72 X10527K2 76 5 75X10528K2 39 3 38 X10529K2 77 10 76 X10530K1 74 8 73 X10531K2 91 10 90X10532K2 75 12 74 X10533K2 91 7 90 X10534K2 76 5 75 X10535K2 65 1 64X10536K2 81 22 80 X10537K1 61 6 60 X10538K1 92 4 91 X10539K2 91 1 90X10540K1 86 2 85 X10541K2 77 4 76 X10542K2 60 3 60 X10543K2 83 7 82X10544K2 38 1 37 X10545K2 64 4 63 X10546K2 54 8 53 X10547K2 94 5 93X10548K2 54 3 53 X10549K1 62 2 61 X10550K2 46 2 45 X10551K1 53 6 52X10552K1 50 6 49 X10553K1 78 8 77 X10554K1 79 9 78 X10555K1 72 6 71X10556K1 76 10 75 X10557K1 68 9 67 X10558K1 64 8 63 X10559K1 68 10 67X10560K1 59 6 58 X10561K1 80 7 78 X10562K1 81 4 80 X10563K1 54 7 52X10564K1 74 4 73 X10565K1 114 5 113 X10566K1 93 11 92 X10567K1 93 10 92X10568K1 85 5 84 X10569K1 58 1 57 X10570K1 78 3 77 X10571K1 86 10 85X10572K1 78 8 77 X10573K1 75 12 74 X10574K1 67 3 65 X10575K1 93 8 92X10576K1 87 7 85 X10577K1 74 8 73 X10578K1 74 7 73 X10579K1 74 6 72X10580K1 61 4 60 X10581K1 109 7 99 X10582K1 114 4 105 X10583K1 117 11108 X10584K1 110 11 101 X10585K1 126 10 117 X10586K1 129 13 120 X10587K1127 10 117 X10588K1 120 18 111 X10589K1 109 7 99 X10590K1 104 10 95X10591K1 106 6 97 X10592K1 112 6 103 X10593K1 91 2 82 X10594K1 75 3 66X10595K1 127 14 118 X10596K1 117 16 108 X10597K2 124 16 115 X10598K1 12113 112 X10599K1 120 10 110 X10600K1 117 10 108 X10601K1 62 2 55 X10602K167 9 59 X10603K1 74 3 67 X10604K1 85 8 77 X10605K1 97 12 90 X10606K1 7710 69 X10607K1 88 11 80 X10608K1 87 7 80 X10609K1 92 12 85 X10610K1 9210 84 X10611K1 71 21 63 X10612K1 84 28 77 X10613K1 88 21 80 X10614K1 9512 87 X10615K1 91 4 84 X10616K1 89 5 82 X10617K1 95 5 87 X10618K1 99 491 X10619K1 89 1 82 X10620K1 85 4 78 X10621K1 87 11 80 X10622K1 83 6 77X10623K1 94 7 87 X10624K1 96 9 90 X10625K1 97 7 91 X10626K1 99 4 92X10627K1 100 9 94 X10628K1 98 7 91 X10629K1 106 8 100 X10630K1 102 7 95

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

1. (canceled)
 2. (canceled)
 3. A single-stranded antisensepolynucleotide agent for inhibiting expression of aminolevulinic acidsynthase-1 (ALAS1), wherein the agent comprises at least 8 contiguousnucleotides differing by no more than 3 nucleotides from any one of thenucleotide sequences listed in Tables 3 and 4, and wherein at least oneof the contiguous nucleotides is a modified nucleotide.
 4. The agent ofclaim 3, wherein substantially all of the nucleotides of the antisensepolynucleotide agent are modified nucleotides.
 5. The agent of claim 3,which is 10 to 40 nucleotides in length; 10 to 30 nucleotides in length;18 to 30 nucleotides in length; or 10 to 24 nucleotides in length. 6.The agent of claim 3, wherein the modified nucleotide comprises amodified sugar moiety selected from the group consisting of: a2′-O-methoxyethyl modified sugar moiety, a 2′-methoxy modified sugarmoiety, a 2′-O-alkyl modified sugar moiety, and a bicyclic sugar moiety.7. The agent of claim 3, wherein the modified nucleotide is a5-methylcytosine.
 8. The agent of claim 3, wherein the modifiednucleotide comprises a modified internucleoside linkage.
 9. The agent ofclaim 3, comprising a plurality of 2′-deoxynucleotides flanked on eachside by at least one nucleotide having a modified sugar moiety.
 10. Theagent of claim 9, wherein the agent is a gapmer comprising a gap segmentcomprised of linked 2′-deoxynucleotides positioned between a 5′ and a 3′wing segment.
 11. The agent of claim 3, comprising a gap segmentconsisting of linked deoxynucleotides; a 5′-wing segment consisting oflinked nucleotides; a 3′-wing segment consisting of linked nucleotides;wherein the gap segment is positioned between the 5′-wing segment andthe 3′-wing segment and wherein each nucleotide of each wing segmentcomprises a modified sugar.
 12. The agent of claim 11, wherein the gapsegment is ten 2′-deoxynucleotides in length and each of the wingsegments is five nucleotides in length.
 13. The agent of claim 3,wherein the agent further comprises a ligand at the 3′-terminus of theagent.
 14. The agent of claim 13, wherein the ligand is anN-acetylgalactosamine (GalNAc) derivative.
 15. A pharmaceuticalcomposition for inhibiting expression of a aminolevulinic acidsynthase-1 (ALAS1) gene comprising the agent of claim
 3. 16. Apharmaceutical composition comprising the agent of claim 3, and a lipidformulation.
 17. A method of inhibiting aminolevulinic acid synthase-1(ALAS1) expression in a cell, the method comprising: (a) contacting thecell with the agent of claim 3; and (b) maintaining the cell produced instep (a) for a time sufficient to obtain antisense inhibition of anALAS1 gene, thereby inhibiting expression of the ALAS gene in the cell.18. The method of claim 17, wherein the cell is within a human subject.19. (canceled)
 20. A method of preventing at least one symptom in ortreating a subject having an ALAS1-associated disease, the methodcomprising administering to the subject a prophylactically effectiveamount or therapeutically effective amount of the agent of claim 3,thereby preventing at least one symptom in or treating the subject. 21.(canceled)
 22. (canceled)
 23. The method of claim 20, wherein theALAS1-associated disease is porphyria.
 24. The method of claim 23,wherein the porphyria is selected from the group consisting of X-linkedsideroblastic anemia (XLSA), ALA deyhdratase deficiency porphyria (Dossporphyria), acute intermittent porphyria (AIP), congenitalerythropoietic porphyria (CEP), prophyria cutanea tarda (PCT),hereditary coproporphyria (coproporphyria, or HCP), variegate porphyria(VP), erythropoietic protoporphyria (EPP), or transient erythroporphyriaof infancy, acute hepatic porphyria, hepatoerythropoietic porphyria, anddual porphyria.
 25. The method of claim 23, wherein the agent or thecomposition is administered to the subject after an acute attack ofporphyria; or during an acute attack of porphyria.